U.S. patent application number 10/560269 was filed with the patent office on 2007-11-08 for antimicrobial peptides.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Hans-Henrik Hogenhaug, Per Holse Mygind, Dorotea Raventos Segura, Alessandro Tossi.
Application Number | 20070259087 10/560269 |
Document ID | / |
Family ID | 33512563 |
Filed Date | 2007-11-08 |
United States Patent
Application |
20070259087 |
Kind Code |
A1 |
Segura; Dorotea Raventos ;
et al. |
November 8, 2007 |
Antimicrobial Peptides
Abstract
The present invention relates to polypeptides having
antimicrobial activity and polynucleotides having a nucleotide
sequence which encodes for the polypeptides. The invention also
relates to nucleic acid constructs, vectors, and host cells
comprising the nucleic acid constructs as well as methods for
producing and using the polypeptides.
Inventors: |
Segura; Dorotea Raventos;
(Humlebaek, DK) ; Mygind; Per Holse; (Soborg,
DK) ; Tossi; Alessandro; (Gorizia, IT) ;
Hogenhaug; Hans-Henrik; (Holte, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
DK-2880
|
Family ID: |
33512563 |
Appl. No.: |
10/560269 |
Filed: |
June 10, 2004 |
PCT Filed: |
June 10, 2004 |
PCT NO: |
PCT/DK04/00399 |
371 Date: |
December 9, 2005 |
Current U.S.
Class: |
426/532 ;
435/320.1; 435/325; 435/71.1; 514/2.3; 514/2.4; 514/2.8; 530/326;
536/22.1; 800/298 |
Current CPC
Class: |
A23K 50/75 20160501;
C11D 3/38 20130101; A61P 31/00 20180101; C07K 14/47 20130101; C07K
14/705 20130101; C07K 7/08 20130101; A23K 50/80 20160501; A61P
31/04 20180101; A23K 50/00 20160501; A23K 20/189 20160501; A23K
20/174 20160501; A23K 20/30 20160501; C07K 14/001 20130101; C11D
3/3719 20130101; A61K 38/00 20130101; C11D 3/48 20130101 |
Class at
Publication: |
426/532 ;
435/320.1; 435/325; 435/071.1; 514/013; 530/326; 536/022.1;
800/298 |
International
Class: |
C07K 14/00 20060101
C07K014/00; A01H 5/00 20060101 A01H005/00; A23K 1/17 20060101
A23K001/17; C07K 14/47 20060101 C07K014/47; C11D 3/00 20060101
C11D003/00; C12N 15/11 20060101 C12N015/11; C07K 7/08 20060101
C07K007/08; A61K 38/16 20060101 A61K038/16; A61P 31/04 20060101
A61P031/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 11, 2003 |
DK |
PA 2003 00865 |
Jun 20, 2003 |
DK |
PA 2003 00924 |
Claims
1. A polypeptide having antimicrobial activity, comprising the
amino acid sequence as set forth in SEQ ID NO:1, or a fragment
thereof of at least 18 amino acids having antimicrobial activity:
G-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-
-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-Z;
Wherein X.sub.1=L, I, W or M; X.sub.2=L, F, W or V; X.sub.3=S, G,
K, T, R, I, N, D or E; X.sub.4=K, T, F, I, R, M, L or S; X.sub.5=L
or I; X.sub.6=K, G, R, M or E; X.sub.7=K, S, I, R, T or M;
X.sub.8=A, K, T, N, R or E; X.sub.9=A, G, S, I, L, T, V, M or W;
X.sub.10=S, R, K or E; X.sub.11=K, M, R, H, I, N or T; X.sub.12=A,
V, I, L, Y, F or T; X.sub.13=L, A, G, C, F, V or W; X.sub.14=K, Q,
A, S, R or E; X.sub.15=H, G, N, R, S, M, I, V or D; X.sub.16=V, I,
A or F; Z=X.sub.17 or X.sub.17-R-W-L; wherein X.sub.17=F, L, R, A,
G, V, Y, C or P; and wherein the amino acids making up the
polypeptide are independently selected from D or L forms.
2. A polypeptide having antimicrobial activity, consisting of an
amino acid sequence which consists of 18 amino acids and which is
extended by the amino acid sequence R--W-L; wherein the amino acids
making up the polypeptide are independently selected from D or L
forms.
3. The polypeptide of claim 1, which comprises the amino acids of
anyone of SEQ ID NO:1 to SEQ ID NO:46.
4. The polypeptide of claim 1, which consists of the amino acids of
anyone of SEQ ID NO:1 to SEQ ID NO:46.
5. A polynucleotide having a nucleotide sequence which encodes for
the polypeptide defined in claim 1.
6. A nucleic acid construct comprising the nucleotide sequence
defined in claim 5 operably linked to one or more control sequences
that direct the production of the polypeptide in a suitable
host.
7. A recombinant expression vector comprising the nucleic acid
construct defined in claim 6.
8. A recombinant host cell comprising the nucleic acid construct
defined in claim 6.
9. A method for producing a polypeptide as defined in claim 1, the
method comprising: (a) cultivating a recombinant host cell as
defined in claim 10 under conditions conducive for production of
the polypeptide; and (b) recovering the polypeptide.
10. A composition comprising an antimicrobial polypeptide as
defined in claim 1.
11. The composition of claim 10, which further comprises an
additional biocidal agent.
12. A method for killing or inhibiting growth of microbial cells
comprising contacting the microbial cells with an antimicrobial
polypeptide as defined in claim 1.
13. A detergent composition comprising a surfactant and an
antimicrobial polypeptide as defined in claim 1.
14. An antimicrobial polypeptide as defined in claim 1 for use as a
medicament.
15. An antimicrobial polypeptide as defined in claim 1 for use as
an antimicrobial veterinarian or human therapeutic or prophylactic
agent.
16. (canceled)
17. (canceled)
18. A transgenic plant, plant part or plant cell, which has been
transformed with a nucleotide sequence encoding a polypeptide
having antimicrobial activity as defined in claim 1.
19. (canceled)
20. (canceled)
21. An animal feed additive comprising (a) at least one
antimicrobial polypeptide as defined in claim 1; and (b) at least
one fat soluble vitamin, and/or (c) at least one water soluble
vitamin, and/or (d) at least one trace mineral, and/or (e) at least
one macro mineral.
22. The animal feed additive of claim 21, which further comprises
phytase, xylanase, galactanase, and/or beta-glucanase.
26. An animal feed composition having a crude protein content of 50
to 800 g/kg and comprising at least one antimicrobial polypeptide
as defined in claim 1.
Description
BACKGROUND
[0001] In recent years, the variety of antimicrobial agents has
increased substantially, along with a parallel increase in
resistant pathogenic microorganisms. Resistance is now recognized
against all clinically available antimicrobial agents. The response
to antimicrobial resistance in the medical community has been to
use new or alternative antibiotics not previously used against the
resistant bacteria. This approach has required the continuous
development of new antibiotics, either as modifications of
currently existing compounds or as combinations of compounds that
may inhibit or bypass the bacterial resistance mechanisms.
[0002] It is an object of the present invention to provide new
polypeptides having improved antimicrobial activity and
polynucleotides encoding the polypeptides.
SUMMARY
[0003] In a first aspect the present invention relates to
polypeptides having antimicrobial activity, comprising the amino
acid sequence, or a fragment thereof of at least 18 amino acids
having antimicrobial activity:
G-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub-
.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16--
Z; wherein X.sub.1=L, I, W or M; X.sub.2=L, F, W or V; X.sub.3=S,
G, K, T, R, I, N, D or E; X.sub.4=K, T, F, I, R, M, L or S;
X.sub.5=L or I; X.sub.6=K, G, R, M or E; X.sub.7=K, S, I, R, T or
M; X.sub.8=A, K, T, N, R or E; X.sub.9=A, G, S, I, L, T, V, M or W;
X.sub.10=S, R, K or E; X.sub.11=K, M, R, H, I, N or T; X.sub.12=A,
V, I, L, Y, F or T; X.sub.13=L, A, G, C, F, V or W; X.sub.14=K, Q,
A, S, R or E; X.sub.15=H, G, N, R, S, M, I, V or D; X.sub.16=V, I,
A or F; Z=X.sub.17 or X.sub.17-R-W-L; wherein X.sub.17=F, L, R, A,
G, V, Y, C or P; and wherein the amino acids making up the
polypeptide are independently selected from D or L forms.
[0004] In another aspect the invention relates to polypeptides
having antimicrobial activity, consisting of an amino acid sequence
which consists of 18 amino acids and which is extended by the amino
acid sequence R-W-L.
[0005] In a second aspect the present invention relates to
polynucleotides having a nucleotide sequence which encodes for the
polypeptide of the invention.
[0006] In a third aspect the present invention relates to a nucleic
acid construct comprising the nucleotide sequence, which encodes
for the polypeptide of the invention, operably linked to one or
more control sequences that direct the production of the
polypeptide in a suitable host.
[0007] In a fourth aspect the present invention relates to a
recombinant expression vector comprising the nucleic acid construct
of the invention.
[0008] In a fifth aspect the present invention relates to a
recombinant host cell comprising the nucleic acid construct of the
Invention.
[0009] In a sixth aspect the present invention relates to a method
for producing a polypeptide of the invention, the method
comprising:
(a) cultivating a recombinant host cell of the invention under
conditions conducive for production of the polypeptide; and
(b) recovering the polypeptide.
[0010] Other aspects of the present invention will be apparent from
the below description and from the appended claims.
Definitions
[0011] Before discussing the present invention in further details,
the following terms and conventions will first be defined:
[0012] Antimicrobial activity: The term "antimicrobial activity" is
defined herein as an activity which is capable of killing or
inhibiting growth of microbial cells. In the context of the present
invention the term "antimicrobial" is intended to mean that there
is a bactericidal and/or a bacteriostatic and/or fungicidal and/or
fungistatic effect and/or a virucidal effect, wherein the term
"bactericidal" 4 is to be understood as capable of killing
bacterial cells. The term "bacteriostatic" is to be understood as
capable of inhibiting bacterial growth, i.e. inhibiting growing
bacterial cells. The term "fungicidal" is to be understood as
capable of killing fungal cells. The term "fungistatic" is to be
understood as capable of inhibiting fungal growth, i.e. inhibiting
growing fungal cells. The term "virucidal" is to be understood as
capable of inactivating virus. The term "microbial cells" denotes
bacterial or fungal cells (including yeasts).
[0013] In the context of the present invention the term "inhibiting
growth of microbial cells" is intended to mean that the cells are
in the non-growing state, i.e., that they are not able to
propagate.
[0014] For purposes of the present invention, antimicrobial
activity may be determined according to the procedure described by
Lehrer et al., Journal of Immunological Methods, Vol. 137 (2) pp.
167-174 (1991).
[0015] Polypeptides having antimicrobial activity may be capable of
reducing the number of living cells of Escherichia coli (DSM 1576)
to 1/100 after 8 hours (preferably after 4 hours, more preferably
after 2 hours, most preferably after 1 hour, and in particular
after 30 minutes) incubation at 20.degree. C. in an aqueous
solution of 25% (w/w); preferably in an aqueous solution of 10%
(w/w); more preferably in an aqueous solution of 5% (w/w); even
more preferably in an aqueous solution of 1% (w/w); most preferably
in an aqueous solution of 0.5% (w/w); and in particular in an
aqueous solution of 0.1% (w/w) of the polypeptides having
antimicrobial activity.
[0016] Polypeptides having antimicrobial activity may also be
capable of inhibiting the outgrowth of Escherichia coli (DSM 1576)
for 24 hours at 25.degree. C. in a microbial growth substrate, when
added in a concentration of 1000 ppm; preferably when added in a
concentration of 500 ppm; more preferably when added in a
concentration of 250 ppm; even more preferably when added in a
concentration of 100 ppm; most preferably when added in a
concentration of 50 ppm; and in particular when added in a
concentration of 25 ppm.
[0017] Polypeptides having antimicrobial activity may be capable of
reducing the number of living cells of Bacillus subtilis (ATCC
6633) to 1/100 after 8 hours (preferably after 4 hours, more
preferably after 2 hours, most preferably after 1 hour, and in
particular after 30 minutes) incubation at 20.degree. C. in an
aqueous solution of 25% (w/w); preferably in an aqueous solution of
10% (w/w); more preferably in an aqueous solution of 5% (w/w); even
more preferably in an aqueous solution of 1% (w/w); most preferably
in an aqueous solution of 0.5% (w/w); and in particular in an
aqueous solution of 0.1% (w/w) of the polypeptides having
antimicrobial activity.
[0018] Polypeptides having antimicrobial activity may also be
capable of inhibiting the outgrowth of Bacillus subtilis (ATCC
6633) for 24 hours at 25.degree. C. in a microbial growth
substrate, when added in a concentration of 1000 ppm; preferably
when added in a concentration of 500 ppm; more preferably when
added in a concentration of 250 ppm; even more preferably when
added in a concentration of 100 ppm; most preferably when added in
a concentration of 50 ppm; and in particular when added in a
concentration of 25 ppm.
[0019] The polypeptides of the present invention should preferably
have at least 20% of the antimicrobial activity of the polypeptide
consisting of the amino acid sequence shown as amino acids 1 to 18
of anyone of SEQ ID NO:1 to SEQ ID NO:37 or amino acids 1 to 21 of
anyone of SEQ ID NO:38 to SEQ ID NO:46. In a particular preferred
embodiment, the polypeptides should have at least 40%, such as at
least 50%, preferably at least 60%, such as at least 70%, more
preferably at least 80%, such as at least 90%, most preferably at
least 95%, such as about or at least 100% of the antimicrobial
activity of the polypeptide consisting of the amino acid sequence
shown as amino acids 1 to 18 of anyone of SEQ ID NO:1 to SEQ ID
NO:37 or amino acids 1 to 21 of anyone of SEQ ID NO:38 to SEQ ID
NO:46.
[0020] Fragment: When used herein, a "fragment" of the amino acid
sequence shown as anyone of SEQ ID NO:1 to SEQ ID NO:46 is a
subsequence of the polypeptides wherein one or more amino acids
have been deleted from the amino and/or carboxyl terminus.
Preferably the one or more amino acids have been deleted from the
carboxyl terminus. More preferably the fragment consists of at
least 18 amino acids.
[0021] Allelic variant: In the present context, the term "allelic
variant" denotes any of two or more alternative forms of a gene
occupying the same chromosomal locus. Allelic variation arises
naturally through mutation, and may result in polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequences. An allelic variant of a polypeptide is a polypeptide
encoded by an allelic variant of a gene.
[0022] Substantially pure polynucleotide: The term "substantially
pure polynucleotide" as used herein refers to a polynucleotide
preparation, wherein the polynucleotide has been removed from its
natural genetic milieu, and is thus free of other extraneous or
unwanted coding sequences and is in a form suitable for use within
genetically engineered protein production systems. Thus, a
substantially pure polynucleotide contains at the most 10% by
weight of other polynucleotide material with which it is natively
associated (lower percentages of other polynucleotide material are
preferred, e.g. at the most 8% by weight, at the most 6% by weight,
at the most 5% by weight, at the most 4% at the most 3% by weight,
at the most 2% by weight, at the most 1% by weight, and at the most
1/2% by weight). A substantially pure polynucleotide may, however,
include naturally occurring 5' and 3' untranslated regions, such as
promoters and terminators. It is preferred that the substantially
pure polynucleotide is at least 92% pure, i.e. that the
polynucleotide constitutes at least 92% by weight of the total
polynucleotide material present in the preparation, and higher
percentages are preferred such as at least 94% pure, at least 95%
pure, at least 96% pure, at least 96% pure, at least 97% pure, at
least 98% pure, at least 99%, and at the most 99.5% pure. The
polynucleotides disclosed herein are preferably in a substantially
pure form. In particular, it is preferred that the polynucleotides
disclosed herein are in "essentially pure form", i.e. that the
polynucleotide preparation is essentially free of other
polynucleotide material with which it is natively associated.
Herein, the term "substantially pure polynucleotide" is synonymous
with the terms "isolated polynucleotide" and "polynucleotide in
isolated form".
[0023] Modification (s): In the context of the present invention
the term "modification(s)" is intended to mean any chemical
modification of the polypeptide consisting of the amino acid
sequence shown as amino acids 1 to 18 of anyone of SEQ ID NO:1 to
SEQ ID NO:37 or amino acids 1 to 21 of anyone of SEQ ID NO:38 to
SEQ ID NO:46 as well as genetic manipulation of the DNA encoding
the polypeptides. The modification(s) can be replacement(s) of the
amino acid side chain(s), substitution(s), deletion(s) and/or
insertions(s) in or at the amino acid(s) of interest; or use of
unnatural amino acids with similar characteristics in the amino
acid sequence. In particular the modification(s) can be amidations,
such as amidation of the C-terminus.
[0024] cDNA: The term "cDNA" when used in the present context, is
intended to cover a DNA molecule which can be prepared by reverse
transcription from a mature, spliced, mRNA molecule derived from a
eukaryotic cell. cDNA lacks the intron sequences that are usually
present in the corresponding genomic DNA. The initial, primary RNA
transcript is a precursor to mRNA and it goes through a series of
processing events before appearing as mature spliced mRNA. These
events include the removal of intron sequences by a process called
splicing. When cDNA is derived from mRNA it therefore lacks intron
sequences.
[0025] Nucleic acid construct: When used herein, the term "nucleic
acid construct" means a nucleic acid molecule, either single- or
double-stranded, which is isolated from a naturally occurring gene
or which has been modified to contain segments of nucleic acids in
a manner that would not otherwise exist in nature. The term nucleic
acid construct is synonymous with the term "expression cassette"
when the nucleic acid construct contains the control sequences
required for expression of a coding sequence of the present
invention.
[0026] Control sequence: The term "control sequences" is defined
herein to include all components, which are necessary or
advantageous for the expression of a polypeptide of the present
invention. Each control sequence may be native or foreign to the
nucleotide sequence encoding the polypeptide. Such control
sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal
peptide sequence, and transcription terminator. At a minimum, the
control sequences include a promoter, and transcriptional and
translational stop signals. The control sequences may be provided
with linkers for the purpose of introducing specific restriction
sites facilitating ligation of the control sequences with the
coding region of the nucleotide sequence encoding a
polypeptide.
[0027] Operably linked: The term "operably linked" is defined
herein as a configuration in which a control sequence is
appropriately placed at a position relative to the coding sequence
of the DNA sequence such that the control sequence directs the
expression of a polypeptide.
[0028] Coding sequence: When used herein the term "coding sequence"
is intended to cover a nucleotide sequence, which directly
specifies the amino acid sequence of its protein product. The
boundaries of the coding sequence are generally determined by an
open reading frame, which usually begins with the ATG start codon.
The coding sequence typically include DNA, cDNA, and recombinant
nucleotide sequences.
[0029] Expression: In the present context, the term "expression"
includes any step involved in the production of the polypeptide
including, but not limited to, transcription, post-transcriptional
modification, translation, and post-translational modification.
Preferably expression also comprise secretion of the
polypeptide.
[0030] Expression vector: In the present context, the term
"expression vector" covers a DNA molecule, linear or circular, that
comprises a segment encoding a polypeptide of the invention, and
which is operably linked to additional segments that provide for
its transcription.
[0031] Host cell: The term "host cell", as used herein, includes
any cell type which is susceptible to transformation with a nucleic
acid construct.
[0032] The terms "polynucleotide probe", "hybridization" as well as
the various stringency conditions are defined in the section titled
"Polypeptides Having Antimicrobial Activity".
DETAILED DESCRIPTION
Polypeptides Having Antimicrobial Activity
[0033] In a first aspect, the present invention relates to
polypeptides having antimicrobial activity and where the
polypeptides comprises, preferably consists of the amino acid
sequence set forth in SEQ ID NO:
G-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-
-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-Z;
wherein X.sub.1=L, I, W or M; X.sub.2=L, F, W or V; X.sub.3=S, G,
K, T, R, I, N, D or E; X.sub.4=K, T, F, I, R, M, L or S; X.sub.5=L
or I; X.sub.6=K, G, R, M or E; X.sub.7=K, S, I, R, T or M;
X.sub.8=A, K, T, N, R or E; X.sub.9=A, G, S, I, L, T, V, M or W;
X.sub.10=S, R, K or E; X.sub.11=K, M, R, H, I, N or T; X.sub.12=A,
V, I, L, Y, F or T; X.sub.13=L, A, G, C, F, V or W; X.sub.14=K, Q,
A, S, R or E; X.sub.15=H, G, N, R, S, M, I, V or D; X.sub.16=V, I,
A or F; Z=X.sub.17 or X.sub.17-R-W-L; wherein X.sub.17=F, L, R, A,
G, V, Y, C or P; or where the polypeptides comprises, preferably
consists of amino acids 1 to 18 of anyone of SEQ ID NO:1 to SEQ ID
NO:37 or amino acids 1 to 21 of anyone of SEQ ID NO:38 to SEQ ID
NO:46. In an interesting embodiment, the amino acid sequence
differs by at the most five amino acids (e.g. by five amino acids),
such as by at the most four amino acids (e.g. by four amino acids),
e.g. by at the most three amino acids (e.g. by three amino acids),
particularly by at the most two amino acids (e.g. by two amino
acids), such as by one amino acid from amino acids 1 to 18 of
anyone of SEQ ID NO:1 to SEQ ID NO:37 or amino acids 1 to 21 of
anyone of SEQ ID NO:38 to SEQ ID NO:46.
[0034] The term "anyone of SEQ ID NO:1 to SEQ ID NO:37" is intended
to mean SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,
SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ
ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,
SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37.
[0035] The term "anyone of SEQ ID NO:38 to SEQ ID NO:46" is
intended to mean SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 or
SEQ ID NO:46.
[0036] Preferably, the polypeptides of the present invention
comprise the amino acid sequence of anyone of SEQ ID NO:1 to SEQ ID
NO:46; or a fragment thereof that has antimicrobial activity. In
another preferred embodiment, the polypeptide of the present
invention comprises amino acids 1 to 18 of anyone of SEQ ID NO:1 to
SEQ ID NO:37 or amino acids 1 to 21 of anyone of SEQ ID NO:38 to
SEQ ID NO:46. In a further preferred embodiment, the polypeptide
consists of amino acids 1 to 18 of anyone of SEQ ID NO:1 to SEQ ID
NO:37 or amino acids 1 to 21 of anyone of SEQ ID NO:38 to SEQ ID
NO:46.
[0037] The amino acids making up the polypeptides of the invention
may independently be selected from D or L forms.
[0038] The polypeptide of the invention may be an artificial
variant which comprises, preferably consists of, an amino acid
sequence that has at the most three, e.g. at the most two, such as
at the most one, substitutions, deletions and/or insertions of
amino acids as compared to amino acids 1 to 18 of anyone of SEQ ID
NO:1 to SEQ ID NO:37 or amino acids 1 to 21 of anyone of SEQ ID
NO:38 to SEQ ID NO:46. Such artificial variants may be constructed
by standard techniques known in the art, such as by
site-directed/random mutagenesis of the polypeptide comprising the
amino acid sequence shown as amino acids 1 to 18 of anyone of SEQ
ID NO:1 to SEQ ID NO:37 or amino acids 1 to 21 of anyone of SEQ ID
NO:38 to SEQ ID NO:46. In one embodiment of the invention, amino
acid changes are of a minor nature, that is conservative amino acid
substitutions that do not significantly affect the folding and/or
activity of the protein; small deletions, typically of one to about
5 amino acids; small amino- or carboxyl-terminal extensions, such
as an amino-terminal methionine residue; a small linker peptide of
up to about 10-25 residues; or a small extension that facilitates
purification by changing net charge or another function, such as a
poly-histidine tract, an antigenic epitope or a binding domain.
[0039] Examples of conservative substitutions are within the group
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine, valine and methionine), aromatic amino acids
(phenylalanine, tryptophan and tyrosine), and small amino acids
(glycine, alanine, serine and threonine). Amino acid substitutions
which do not generally alter the specific activity are known in the
art and are described, for example, by H. Neurath and R. L. Hill,
1979, In, The Proteins, Academic Press, New York. The most commonly
occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,
Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro,
Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as
these in reverse.
[0040] In an interesting embodiment of the invention, the amino
acid changes are of such a nature that the physico-chemical
properties of the polypeptides are altered. For example, amino acid
changes may be performed, which improve the thermal stability of
the polypeptide, which alter the substrate specificity, which
changes the pH optimum, and the like.
[0041] In another aspect, the invention relates to polypeptides
having antimicrobial activity, consisting of an amino acid sequence
which consists of 18 or 19 amino acids and which is extended by the
amino acid sequence R-W-L. In an embodiment the amino acid sequence
consists of 18 amino acids. In another embodiment the first amino
acid of the amino acid sequence is glycine.
N-Terminal Extension
[0042] An N-terminal extension of the polypeptides of the invention
may suitably consist of from 1 to 50 amino acids, preferably 2-20
amino acids, especially 3-15 amino acids. In one embodiment
N-terminal peptide extension does not contain an Arg (R). In
another embodiment the N-terminal extension comprises a kex2 or
kex2-like cleavage site as will be defined further below. In a
preferred embodiment the N-terminal extension is a peptide,
comprising at least two Glu (E) and/or Asp (D) amino acid residues,
such as an N-terminal extension comprising one of the following
sequences: EAE, EE, DE and DD.
Kex2 Sites
[0043] Kex2 sites (see, e.g., Methods in Enzymology Vol 185, ed. D.
Goeddel, Academic Press Inc. (1990), San Diego, Calif., "Gene
Expression Technology") and kex2-like sites are di-basic
recognition sites (i.e., cleavage sites), found between the
pro-peptide encoding region and the mature region of some
proteins.
[0044] Insertion of a kex2 site or a kex2-like site have in certain
cases been shown to improve correct endopeptidase processing at the
pro-peptide cleavage site resulting in increased protein secretion
levels.
[0045] In the context of the invention insertion of a kex2 or
kex2-like site result in the possibility to obtain cleavage at a
certain position in the N-terminal extension resulting in an
antimicrobial polypeptide being extended in comparison to the
mature polypeptide shown as amino acids 1 to 18 of anyone of SEQ ID
NO:1 to SEQ ID NO:37 or amino acids 1 to 21 of anyone of SEQ ID
NO:38 to SEQ ID NO:46.
[0046] Fused Polypeptides
[0047] The polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide of the invention or a fragment thereof. A fused
polypeptide is produced by fusing a nucleotide sequence (or a
portion thereof) encoding another polypeptide to a nucleotide
sequence (or a portion thereof) of the present invention.
Techniques for producing fusion polypeptides are known in the art,
and include ligating the coding sequences encoding the polypeptides
so that they are in frame and that expression of the fused
polypeptide is under control of the same promoter(s) and
terminator.
Polynucleotides and Nucleotide Sequences
[0048] The present invention also relates to polynucleotides having
a nucleotide sequence which encodes for a polypeptide of the
invention. In particular, the present invention relates to
polynucleotides consisting of a nucleotide sequence which encodes
for a polypeptide of the invention. Due to the degeneracy of the
genetic code, the skilled person will easily recognize that several
nucleotide sequences encoding each of the polypeptides of the
invention may be prepared. It is well known in the art which
nucleotides make up codons encoding the amino acids of the
polypeptides of the invention.
[0049] The present invention also relates to polynucleotides which
encode fragments of the amino acid sequence shown as anyone of SEQ
ID NO:1 to SEQ ID NO:46 that have antimicrobial activity. A
subsequence of the polynucleotides is a nucleotide sequence wherein
one or more nucleotides from the 5' and/or 3' end have been
deleted.
[0050] The nucleotide sequence may be obtained by standard cloning
procedures used in genetic engineering to relocate the nucleotide
sequence from one location to a different site where it will be
reproduced. The cloning procedures may involve excision and
isolation of a desired fragment comprising the nucleotide sequence
encoding the polypeptide, insertion of the fragment into a vector
molecule, and incorporation of the recombinant vector into a host
cell where multiple copies or clones of the nucleotide sequence
will be replicated. The nucleotide sequence may be of genomic,
cDNA, RNA, semisynthetic, synthetic origin, or any combinations
thereof.
[0051] Modification of a nucleotide sequence encoding a polypeptide
of the present invention may be necessary for the synthesis of a
polypeptide, which comprises an amino acid sequence that has at
least one substitution, deletion and/or insertion as compared to
amino acids 1 to 18 of anyone of SEQ ID NO:1 to SEQ ID NO:37 or
amino acids 1 to 21 of anyone of SEQ ID NO:38 to SEQ ID NO:46.
These artificial variants may differ in some engineered way from
the polypeptide isolated from its native source, e.g., variants
that differ in specific activity, thermostability, pH optimum, or
the like.
[0052] It will be apparent to those skilled in the art that such
modifications can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acid residues essential to the activity of the polypeptide
encoded by the nucleotide sequence of the invention, and therefore
preferably not subject to modification, such as substitution, may
be identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis (see,
e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the
latter technique, mutations are introduced at every positively
charged residue in the molecule, and the resultant mutant molecules
are tested for antimicrobial activity to identify amino acid
residues that are critical to the activity of the molecule. Sites
of substrate-enzyme interaction can also be determined by analysis
of the three-dimensional structure as determined by such techniques
as nuclear magnetic resonance analysis, crystallography or
photoaffinity labelling (see, e.g., de Vos et al., 1992, Science
255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224:
899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
[0053] Moreover, a nucleotide sequence encoding a polypeptide of
the present invention may be modified by introduction of nucleotide
substitutions which do not give rise to another amino acid sequence
of the polypeptide encoded by the nucleotide sequence, but which
correspond to the codon usage of the host organism intended for
production of the enzyme.
[0054] The introduction of a mutation Into the nucleotide sequence
to exchange one nucleotide for another nucleotide may be
accomplished by site-directed mutagenesis using any of the methods
known in the art. Particularly useful is the procedure, which
utilizes a supercoiled, double stranded DNA vector with an insert
of interest and two synthetic primers containing the desired
mutation. The oligonucleotide primers, each complementary to
opposite strands of the vector, extend during temperature cycling
by means of Pfu DNA polymerase. On incorporation of the primers, a
mutated plasmid containing staggered nicks is generated. Following
temperature cycling, the product is treated with DpnI which is
specific for methylated and hemimethylated DNA to digest the
parental DNA template and to select for mutation-containing
synthesized DNA. Other procedures known in the art may also be
used. For a general description of nucleotide substitution, see,
e.g., Ford et al., 1991, Protein Expression and Purification 2:
95-107.
Nucleic Acid Constructs
[0055] The present invention also relates to nucleic acid
constructs comprising a nucleotide sequence of the present
invention operably linked to one or more control sequences that
direct the expression of the coding sequence in a suitable host
cell under conditions compatible with the control sequences.
[0056] A nucleotide sequence encoding a polypeptide of the present
invention may be manipulated in a variety of ways to provide for
expression of the polypeptide. Manipulation of the nucleotide
sequence prior to its insertion into a vector may be desirable or
necessary depending on the expression vector. The techniques for
modifying nucleotide sequences utilizing recombinant DNA methods
are well known in the art.
[0057] The control sequence may be an appropriate promoter
sequence, a nucleotide sequence which is recognized by a host cell
for expression of the nucleotide sequence. The promoter sequence
contains transcriptional control sequences, which mediate the
expression of the polypeptide. The promoter may be any nucleotide
sequence which shows transcriptional activity in the host cell of
choice including mutant, truncated, and hybrid promoters, and may
be obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host
cell.
[0058] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention, especially in a bacterial host cell, are the promoters
obtained from the E. coli lac operon, Streptomyces coelicolor
agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),
Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
penicillinase gene (penP), Bacillus subtilis xyla and xylB genes,
and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 3727-3731),
as well as the tac promoter (DeBoer et al., 1983, Proceedings of
the National Academy of Sciences USA 80: 21-25). Further promoters
are described in "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242: 74-94; and in Sambrook et al.,
1989, supra.
[0059] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, and Fusarium oxysporum trypsin-like protease (WO
96/00787), as well as the NA2-tpi promoter (a hybrid of the
promoters from the genes for Aspergillus niger neutral
alpha-amylase and Aspergillus oryzae triose phosphate isomerase),
and mutant, truncated, and hybrid promoters thereof.
[0060] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP),
and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other
useful promoters for yeast host cells are described by Romanos et
al., 1992, Yeast 8: 423-488.
[0061] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any terminator which is functional in the host cell of
choice may be used in the present invention.
[0062] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0063] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C(CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0064] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA which is important for
translation by the host cell. The leader sequence is operably
linked to the 5' terminus of the nucleotide sequence encoding the
polypeptide. Any leader sequence that is functional in the host
cell of choice may be used in the present invention.
[0065] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0066] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0067] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleotide
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence which is functional in the host cell
of choice may be used in the present invention.
[0068] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0069] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0070] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleotide sequence may inherently contain a signal peptide
coding region naturally linked in translation reading frame with
the segment of the coding region which encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region which is foreign to the
coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a
signal peptide coding region. Alternatively, the foreign signal
peptide coding region may simply replace the natural signal peptide
coding region in order to enhance secretion of the polypeptide.
However, any signal peptide coding region which directs the
expressed polypeptide into the secretory pathway of a host cell of
choice may be used in the present invention.
[0071] Effective signal peptide coding regions for bacterial host
cells are the signal peptide coding regions obtained from the genes
for Bacillus NCIB 11837 maltogenic amylase, Bacillus
stearothermophilus alpha-amylase, Bacillus licheniformis
subtilisin, Bacillus licheniformis beta-lactamase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0072] Effective signal peptide coding regions for filamentous
fungal host cells are the signal peptide coding regions obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, and
Humicola lanuginosa lipase.
[0073] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding regions are described by Romanos et al., 1992, supra.
[0074] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a polypeptide. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypepide is generally inactive and can be converted to a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Bacillus subtilis
alkaline protease (aprE), Bacillus subtilis neutral protease
(nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei
aspartic proteinase, and Myceliophthora thermophila laccase (WO
95/33836).
[0075] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0076] It may also be desirable to add regulatory sequences which
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those which cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GALL system may be used. In
filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus
niger glucoamylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used as regulatory sequences. Other examples of
regulatory sequences are those which allow for gene amplification.
In eukaryotic systems, these include the dihydrofolate reductase
gene which is amplified in the presence of methotrexate, and the
metallothionein genes which are amplified with heavy metals. In
these cases, the nucleotide sequence encoding the polypeptide would
be operably linked with the regulatory sequence.
Expression Vectors
[0077] The present invention also relates to recombinant expression
vectors comprising the nucleic acid construct of the invention. The
various nucleotide and control sequences described above may be
joined together to produce a recombinant expression vector which
may include one or more convenient restriction sites to allow for
insertion or substitution of the nucleotide sequence encoding the
polypeptide at such sites. Alternatively, the nucleotide sequence
of the present invention may be expressed by inserting the
nucleotide sequence or a nucleic acid construct comprising the
sequence into an appropriate vector for expression. In creating the
expression vector, the coding sequence is located in the vector so
that the coding sequence is operably linked with the appropriate
control sequences for expression.
[0078] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to
recombinant DNA procedures and can bring about the expression of
the nucleotide sequence. The choice of the vector will typically
depend on the compatibility of the vector with the host cell into
which the vector is to be introduced. The vectors may be linear or
closed circular plasmids.
[0079] The vector may be an autonomously replicating vector, i.e.,
a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome.
[0080] The vector may contain any means for assuring
self-replication. Alternatively, the vector may be one which, when
introduced into the host cell, is integrated into the genome and
replicated together with the chromosome(s) into which it has been
integrated. Furthermore, a single vector or plasmid or two or more
vectors or plasmids which together contain the total DNA to be
introduced into the genome of the host cell, or a transposon may be
used.
[0081] The vectors of the present invention preferably contain one
or more selectable markers which permit easy selection of
transformed cells. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs, and the like.
[0082] Examples of bacterial selectable markers are the daI genes
from Bacillus subtilis or Bacillus licheniformis, or markers which
confer antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or tetracycline resistance. Suitable markers for
yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hygB (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), trpC (anthranilate synthase), as
well as equivalents thereof.
[0083] Preferred for use in an Aspergillus cell are the amdS and
pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the
bar gene of Streptomyces hygroscopicus.
[0084] The vectors of the present invention preferably contain an
element(s) that permits stable integration of the vector into the
host cell's genome or autonomous replication of the vector in the
cell independent of the genome.
[0085] For integration into the host cell genome, the vector may
rely on the nucleotide sequence encoding the polypeptide or any
other element of the vector for stable integration of the vector
into the genome by homologous or nonhomologous recombination.
Alternatively, the vector may contain additional nucleotide
sequences for directing integration by homologous recombination
into the genome of the host cell. The additional nucleotide
sequences enable the vector to be integrated into the host cell
genome at a precise location(s) in the chromosome(s). To increase
the likelihood of integration at a precise location, the
integrational elements should preferably contain a sufficient
number of nucleotides, such as 100 to 1,500 base pairs, preferably
400 to 1,500 base pairs, and most preferably 800 to 1,500 base
pairs, which are highly homologous with the corresponding target
sequence to enhance the probability of homologous recombination.
The integrational elements may be any sequence that is homologous
with the target sequence in the genome of the host cell.
Furthermore, the integrational elements may be non-encoding or
encoding nucleotide sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0086] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. Examples of bacterial
origins of replication are the origins of replication of plasmids
pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E
coli, and pUB110, pE194, pTA1060, and pAM.beta.1 permitting
replication in Bacillus. Examples of origins of replication for use
in a yeast host cell are the 2 micron origin of replication, ARS1,
ARS4, the combination of ARS1 and CEN3, and the combination of ARS4
and CEN6. The origin of replication may be one having a mutation
which makes its functioning temperature-sensitive in the host cell
(see, e.g., Ehrlich, 1978, Proceedings of the National Academy of
Sciences USA 75: 1433).
[0087] More than one copy of a nucleotide sequence of the present
invention may be inserted into the host cell to increase production
of the gene product. An increase in the copy number of the
nucleotide sequence can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the nucleotide
sequence where cells containing amplified copies of the selectable
marker gene, and thereby additional copies of the nucleotide
sequence, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0088] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0089] The present invention also relates to recombinant a host
cell comprising the nucleic acid construct of the invention, which
are advantageously used in the recombinant production of the
polypeptides. A vector comprising a nucleotide sequence of the
present invention is introduced into a host cell so that the vector
is maintained as a chromosomal integrant or as a self-replicating
extra-chromosomal vector as described earlier.
[0090] The host cell may be a unicellular microorganism, e.g., a
prokaryote, or a non-unicellular microorganism, e.g., a
eukaryote.
[0091] Useful unicellular cells are bacterial cells such as gram
positive bacteria including, but not limited to, a Bacillus cell,
e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus
brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,
Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus stearothermophilus, Bacillus subtilis, and
Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces
lividans or Streptomyces murinus, or gram negative bacteria such as
E. coli and Pseudomonas sp. In a preferred embodiment, the
bacterial host cell is a Bacillus lentus, Bacillus licheniformis,
Bacillus stearothermophilus, or Bacillus subtilis cell. In another
preferred embodiment, the Bacillus cell is an alkalophilic
Bacillus.
[0092] The introduction of a vector into a bacterial host cell may,
for instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Molecular General Genetics 168: 111-115),
using competent cells (see, e.g., Young and Spizizin, 1961, Journal
of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971,
Journal of Molecular Biology 56: 209-221), electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169: 5771-5278).
[0093] The host cell may be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0094] In a preferred embodiment, the host cell is a fungal cell.
"Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et
al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et
al., 1995, supra).
[0095] In a more preferred embodiment, the fungal host cell is a
yeast cell. "Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0096] In an even more preferred embodiment, the yeast host cell is
a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell.
[0097] In a most preferred embodiment, the yeast host cell is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
In another most preferred embodiment, the yeast host cell is a
Kluyveromyces lactis cell. In another most preferred embodiment,
the yeast host cell is a Yarrowia lipolytica cell.
[0098] In another more preferred embodiment, the fungal host cell
is a filamentous fungal cell. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are characterized by a mycelial wall composed of chitin, cellulose,
glucan, chitosan, mannan, and other complex polysaccharides.
Vegetative growth is by hyphal elongation and carbon catabolism is
obligately aerobic. In contrast, vegetative growth by yeasts such
as Saccharomyces cerevisiae is by budding of a unicellular thallus
and carbon catabolism may be fermentative.
[0099] In an even more preferred embodiment, the filamentous fungal
host cell is a cell of a species of, but not limited to,
Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora,
Neurospora, Penicillium, Thielavia, Tolypocladium, or
Trichoderma.
[0100] In a most preferred embodiment, the filamentous fungal host
cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus
japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus
oryzae cell. In another most preferred embodiment, the filamentous
fungal host cell is a Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
or Fusarium venenatum cell. In an even most preferred embodiment,
the filamentous fungal parent cell is a Fusarium venenatum
(Nirenberg sp. nov.) cell. In another most preferred embodiment,
the filamentous fungal host cell is a Humicola insolens, Humicola
lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora
crassa, Penicillum purpurogenum, Thielavia terrestris, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, or Trichoderma viride cell.
[0101] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus host cells are
described in EP 238 023 and Yelton et al., 1984, Proceedings of the
National Academy of Sciences USA 81: 1470-1474. Suitable methods
for transforming Fusarium species are described by Malardier et
al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be
transformed using the procedures described by Becker and Guarente,
In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast
Genetics and Molecular Biology, Methods in Enzymology, Volume 194,
pp 182-187, Academic Press, Inc., New York; Ito et al., 1983,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 1920.
Methods of Production
[0102] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
host cell under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0103] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the polypeptide using methods known in the art, For example, the
cell may be cultivated by shake flask cultivation, small-scale or
large-scale fermentation (including continuous, batch, fed-batch,
or solid state fermentations) in laboratory or industrial
fermentors performed in a suitable medium and under conditions
allowing the polypeptide to be expressed and/or isolated. The
cultivation takes place in a suitable nutrient medium comprising
carbon and nitrogen sources and inorganic salts, using procedures
known in the art. Suitable media are available from commercial
suppliers or may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection). If
the polypeptide is secreted into the nutrient medium, the
polypeptide can be recovered directly from the medium. If the
polypeptide is not secreted, it can be recovered from cell
lysates.
[0104] The polypeptides may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include use of specific antibodies, formation of an enzyme
product, or disappearance of an enzyme substrate. For example, an
enzyme assay may be used to determine the activity of the
polypeptide as described herein.
[0105] The resulting polypeptide may be recovered by methods known
in the art. For example, the polypeptide may be recovered from the
nutrient medium by conventional procedures including, but not
limited to, centrifugation, filtration, extraction, spray-drying,
evaporation, or precipitation.
[0106] The polypeptides of the present invention may be purified by
a variety of procedures known in the art including, but not limited
to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction
(see, e.g., Protein Purification, J.-C. Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989).
Plants
[0107] The present invention also relates to a transgenic plant,
plant part, or plant cell which has been transformed with a
nucleotide sequence encoding a polypeptide having antimicrobial
activity of the present invention so as to express and produce the
polypeptide in recoverable quantities. The polypeptide may be
recovered from the plant or plant part. Alternatively, the plant or
plant part containing the recombinant polypeptide may be used as
such for improving the quality of a food or feed, e.g., improving
nutritional value, palatability, and rheological properties, or to
destroy an antinutritive factor. The recovered polypeptide, plant
or plant part may also be used to improve or alter digestive flora
in animals and livestock.
[0108] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0109] Examples of dicot plants are tobacco, potato, sugar beet,
legumes, such as lupins, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0110] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers. Also specific plant tissues, such as
chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and
cytoplasm are considered to be a plant part. Furthermore, any plant
cell, whatever the tissue origin, is considered to be a plant
part.
[0111] Also included within the scope of the present invention are
the progeny of such plants, plant parts and plant cells.
[0112] The transgenic plant or plant cell expressing a polypeptide
of the present invention may be constructed in accordance with
methods known in the art. Briefly, the plant or plant cell is
constructed by incorporating one or more expression constructs
encoding a polypeptide of the present invention into the plant host
genome and propagating the resulting modified plant or plant cell
into a transgenic plant or plant cell.
[0113] Conveniently, the expression construct is a nucleic acid
construct which comprises a nucleotide sequence encoding a
polypeptide of the present invention operably linked with
appropriate regulatory sequences required for expression of the
nucleotide sequence in the plant or plant part of choice.
Furthermore, the expression construct may comprise a selectable
marker useful for identifying host cells into which the expression
construct has been integrated and DNA sequences necessary for
introduction of the construct into the plant in question (the
latter depends on the DNA introduction method to be used).
[0114] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences is
determined, for example, on the basis of when, where, and how the
polypeptide is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide of the present
invention may be constitutive or inducible, or may be
developmental, stage or tissue specific, and the gene product may
be targeted to a specific tissue or plant part such as seeds or
leaves. Regulatory sequences are, for example, described by Tague
et al., 1988, Plant Physiology 86: 506.
[0115] For constitutive expression, the 35S-CaMV promoter may be
used (Franck et al., 1980, Cell 21: 285-294). Organ-specific
promoters may be, for example, a promoter from storage sink tissues
such as seeds, potato tubers, and fruits (Edwards & Coruzzi,
1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues
such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878),
a seed specific promoter such as the glutelin, prolamin, globulin,
or albumin promoter from rice (Wu et al., 1998, Plant and Cell
Physiology 39: 885-889), a Vicia faba promoter from the legumin B4
and the unknown seed protein gene from Vicia faba (Conrad et al.,
1998, Journal of Plant Physiology 152: 708-711), a promoter from a
seed oil body protein (Chen et al., 1998, Plant and Cell Physiology
39: 935-941), the storage protein napA promoter from Brassica
napus, or any other seed specific promoter known in the art, e.g.,
as described in WO 91/14772. Furthermore, the promoter may be a
leaf specific promoter such as the rbcs promoter from rice or
tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the
chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene
promoter from rice (Kagaya et al., 1995, Molecular and General
Genetics 248: 668-674), or a wound inducible promoter such as the
potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22:
573-588).
[0116] A promoter enhancer element may also be used to achieve
higher expression of the enzyme in the plant. For instance, the
promoter enhancer element may be an intron which is placed between
the promoter and the nucleotide sequence encoding a polypeptide of
the present invention. For instance, Xu et al., 1993, supra
disclose the use of the first intron of the rice actin 1 gene to
enhance expression.
[0117] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0118] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0119] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology
19: 15-38). However it can also be used for transforming monocots,
although other transformation methods are generally preferred for
these plants. Presently, the method of choice for generating
transgenic monocots is particle bombardment (microscopic gold or
tungsten particles coated with the transforming DNA) of embryonic
calli or developing embryos (Christou, 1992, Plant Journal 2:
275-281; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162;
Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative
method for transformation of monocots is based on protoplast
transformation as described by Omirulleh et al., 1993, Plant
Molecular Biology 21: 415-428.
[0120] Following transformation, the transformants having
incorporated therein the expression construct are selected and
regenerated into whole plants according to methods well-known in
the art.
[0121] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
transgenic plant or a plant cell comprising a nucleotide sequence
encoding a polypeptide having antimicrobial activity of the present
invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
Compositions
[0122] In a still further aspect, the present invention relates to
compositions, such as pharmaceutical compositions, comprising an
antimicrobial polypeptide of the invention.
[0123] The composition may comprise a polypeptide of the invention
as the major polypeptide component, e.g., a mono-component
composition. Alternatively, the composition may comprise multiple
enzymatic activities, such as an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,
esterase, alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase,
laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,
peptidoglutaminase, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or
xylanase.
[0124] The compositions may further comprise another
pharmaceutically active agent, such as an additional biocidal
agent, such as another antimicrobial polypeptide exhibiting
antimicrobial activity as defined above. The biocidal agent may be
an antibiotic, as known in the art. Classes of antibiotics include
penicillins, e.g. penicillin G, penicillin V, methicillin,
oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins
in combination with beta-lactamase inhibitors, cephalosporins, e.g.
cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems;
monobactams; aminoglycosides; tetracyclines; macrolides;
lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical;
metronidazole; spectinomycin; trimethoprim; vancomycin; etc. The
biocidal agent may also be an anti-mycotic agent, including
polyenes, e.g. amphotericin B, nystatin; 5-flucosyn; and azoles,
e.g. miconazol, ketoconazol, itraconazol and fluconazol.
[0125] In an embodiment the biocidal agent is a non-enzymatic
chemical agent. In another embodiment the biocidal agent is a
non-polypeptide chemical agent.
[0126] The biocidal agent may be capable of reducing the number of
living cells of Escherichia coli (DSM 1576) to 1/100 after 8 hours
(preferably after 4 hours, more preferably after 2 hours, most
preferably after 1 hour, and in particular after 30 minutes)
incubation at 20.degree. C. in an aqueous solution of 25% (w/w);
preferably in an aqueous solution of 10% (w/w); more preferably in
an aqueous solution of 5% (w/w); even more preferably in an aqueous
solution of 1% (w/w); most preferably in an aqueous solution of
0.5% (w/w); and in particular in an aqueous solution of 0.1% (w/w)
of the biocidal agent.
[0127] The biocidal agent may also be capable of inhibiting the
outgrowth of Escherichia coli (DSM 1576) for 24 hours at 25.degree.
C. in a microbial growth substrate, when added in a concentration
of 1000 ppm; preferably when added in a concentration of 500 ppm;
more preferably when added in a concentration of 250 ppm; even more
preferably when added in a concentration of 100 ppm; most
preferably when added in a concentration of 50 ppm; and in
particular when added in a concentration of 25 ppm.
[0128] The biocidal agent may also be capable of reducing the
number of living cells of Bacillus subtilis (ATCC 6633) to 1/100
after 8 hours (preferably after 4 hours, more preferably after 2
hours, most preferably after 1 hour, and in particular after 30
minutes) incubation at 20.degree. C. in an aqueous solution of 25%
(w/w); preferably in an aqueous solution of 10% (w/w); more
preferably in an aqueous solution of 5% (w/w); even more preferably
in an aqueous solution of 1% (w/w); most preferably in an aqueous
solution of 0.5% (w/w); and in particular in an aqueous solution of
0.1% (w/w) of the biocidal agent.
[0129] The biocidal agent may also be capable of inhibiting the
outgrowth of Bacillus subtilis (ATCC 6633) for 24 hours at
25.degree. C. in a microbial growth substrate, when added in a
concentration of 1000 ppm; preferably when added in a concentration
of 500 ppm; more preferably when added in a concentration of 250
ppm; even more preferably when added in a concentration of 100 ppm;
most preferably when added in a concentration of 50 ppm; and in
particular when added in a concentration of 25 ppm.
[0130] The compositions may comprise a suitable carrier material.
The compositions may also comprise a suitable delivery vehicle
capable of delivering the antimicrobial polypeptides of the
invention to the desired locus when the compositions are used as a
medicament.
[0131] The compositions may be prepared in accordance with methods
known in the art and may be in the form of a liquid or a dry
composition. For instance, the polypeptide composition may be in
the form of a granulate or a microgranulate. The polypeptide to be
included in the composition may be stabilized in accordance with
methods known in the art.
[0132] Examples are given below of preferred uses of the
polypeptide compositions of the invention. The dosage of the
polypeptide composition of the invention and other conditions under
which the composition is used may be determined on the basis of
methods known in the art.
Methods and Uses
[0133] The present invention also encompasses various uses of the
antimicrobial polypeptides of the invention. The antimicrobial
polypeptides are typically useful at any locus subject to
contamination by bacteria, fungi, yeast or algae. Typically, loci
are in aqueous systems such as cooling water systems, laundry rinse
water, oil systems such as cutting oils, lubricants, oil fields and
the like, where microorganisms need to be killed or where their
growth needs to be controlled. However, the present invention may
also be used in all applications for which known antimicrobial
compositions are useful, such as protection of wood, latex,
adhesive, glue, paper, cardboard, textile, leather, plastics,
caulking, and feed.
[0134] Other uses include preservation of foods, beverages,
cosmetics such as lotions, creams, gels, ointments, soaps,
shampoos, conditioners, antiperspirants, deodorants, mouth wash,
contact lens products, enzyme formulations, or food
ingredients.
[0135] Thus, the antimicrobial polypeptides of the invention may by
useful as a disinfectant, e.g., in the treatment of acne,
infections in the eye or the mouth, skin infections; in
antiperspirants or deodorants; in foot bath salts; for cleaning and
disinfection of contact lenses, hard surfaces, teeth (oral care),
wounds, bruises and the like.
[0136] In general it is contemplated that the antimicrobial
polypeptides of the present invention are useful for cleaning,
disinfecting or inhibiting microbial growth on any hard surface.
Examples of surfaces, which may advantageously be contacted with
the antimicrobial polypeptides of the invention are surfaces of
process equipment used e.g. dairies, chemical or pharmaceutical
process plants, water sanitation systems, oil processing plants,
paper pulp processing plants, water treatment plants, and cooling
towers. The antimicrobial polypeptides of the invention should be
used in an amount, which is effective for cleaning, disinfecting or
inhibiting microbial growth on the surface in question.
[0137] Further, it is contemplated that the antimicrobial
polypeptides of the invention can advantageously be used in a
cleaning-in-place (C.I.P.) system for cleaning of process equipment
of any kind.
[0138] The antimicrobial polypeptides of the invention may
additionally be used for cleaning surfaces and cooking utensils in
food processing plants and in any area in which food is prepared or
served such as hospitals, nursing homes, restaurants, especially
fast food restaurants, delicatessens and the like. It may also be
used as an antimicrobial in food products and would be especially
useful as a surface antimicrobial in cheeses, fruits and vegetables
and food on salad bars.
[0139] It may also be used as a preservation agent or a
disinfection agent in water based paints.
[0140] The antimicrobial polypeptides of the present invention are
also useful for microbial control of water lines, and for
disinfection of water, in particular for disinfection of industrial
water.
[0141] The invention also relates to the use of an antimicrobial
polypeptide or composition of the invention as a medicament.
Further, an antimicrobial polypeptide or composition of the
invention may also be used for the manufacture of a medicament for
controlling or combating microorganisms, such as fungal organisms
or bacteria, preferably gram positive bacteria.
[0142] The composition and antimicrobial polypeptide of the
invention may be used as an antimicrobial veterinarian or human
therapeutic or prophylactic agent. Thus, the composition and
antimicrobial polypeptide of the invention may be used in the
preparation of veterinarian or human therapeutic agents or
prophylactic agents for the treatment of microbial infections, such
as bacterial or fungal infections, preferably gram positive
bacterial infections. In particular the microbial infections may be
associated with lung diseases including, but not limited to,
tuberculosis, pneumonia and cystic fibrosis; and sexual transmitted
diseases including, but not limited to, gonorrhea and
chlamydia.
[0143] The composition of the invention comprises an effective
amount of the antimicrobial polypeptide of the invention.
[0144] The term "effective amount" when used herein is intended to
mean an amount of the antimicrobial polypeptide comprising the
amino acid sequence shown as amino acids 1 to 18 of anyone of SEQ
ID NO:1 to SEQ ID NO:37 or amino acids 1 to 21 of anyone of SEQ ID
NO:38 to SEQ ID NO:46, or a fragment or a variant thereof, which is
sufficient to inhibit growth of the microorganisms in question.
[0145] The invention also relates to wound healing compositions or
products such as bandages, medical devices such as, e.g., catheters
and further to anti-dandruff hair products, such as shampoos.
[0146] Formulations of the antimicrobial polypeptides of the
invention are administered to a host suffering from or predisposed
to a microbial infection. Administration may be topical, localized
or systemic, depending on the specific microorganism, preferably it
will be localized. Generally the dose of the antimicrobial
polypeptides of the invention will be sufficient to decrease the
microbial population by at least about 50%, usually by at least 1
log, and may be by 2 or more logs of killing. The compounds of the
present invention are administered at a dosage that reduces the
microbial population while minimizing any side-effects. It is
contemplated that the composition will be obtained and used under
the guidance of a physician for in vivo use. The antimicrobial
polypeptides of the invention are particularly useful for killing
gram negative bacteria, including Pseudomonas aeruginosa, and
Chlamydia trachomatis; and gram-positive bacteria, including
various staphylococci and streptococci.
[0147] The antimicrobial polypeptides of the invention are also
useful for in vitro formulations to kill microbes, particularly
where one does not wish to introduce quantities of conventional
antibiotics. For example, the antimicrobial polypeptides of the
invention may be added to animal and/or human food preparations; or
they may be included as an additive for in vitro cultures of cells,
to prevent the overgrowth of microbes in tissue culture.
[0148] The susceptibility of a particular microbe to killing with
the antimicrobial polypeptides of the invention may be determined
by in vitro testing, as detailed in the experimental section.
Typically a culture of the microbe is combined with the
antimicrobial polypeptide at varying concentrations for a period of
time sufficient to allow the protein to act, usually between about
one hour and one day. The viable microbes are then counted, and the
level of killing determined.
[0149] Microbes of interest include, but are not limited to,
Gram-negative bacteria, for example: Citrobacter sp.; Enterobacter
sp.; Escherichia sp., e.g. E. coli; Klebsiella sp.; Morganella sp.;
Proteus sp.; Providencia sp.; Salmonella sp., e.g. S. typhi, S.
typhimurium; Serratia sp.; Shigella sp.; Pseudomonas sp., e.g. P.
aeruginosa; Yersinia sp., e.g. Y. pestis, Y. pseudotuberculosis, Y.
enterocolitica; Franciscella sp.; Pasturella sp.; Vibrio sp., e.g.
V. cholerae, V. parahemolyticus; Campylobacter sp., e.g. C. jejuni,
Haemophilus sp., e.g. H. influenzae, H. ducreyi; Bordetella sp.,
e.g. B. pertussis, B. bronchiseptica, B. parapertussis; Brucella
sp., Neisseria sp., e.g. N. gonorrhoeae, N. meningitidis, etc.
Other bacteria of interest include Legionella sp., e.g. L.
pneumophila; Listeria sp., e.g. L. monocytogenes; Mycoplasma sp.,
e.g. M. hominis, M. pneumoniae; Mycobacterium sp., e.g. M.
tuberculosis, M. leprae; Treponema sp., e.g. T. pallidum; Borrelia
sp., e.g. B. burgdorferi; Leptospirae sp.; Rickettsia sp., e.g. R.
rickettsii, R. typhi; Chlamydia sp., e.g. C. trachomatis, C.
pneumoniae, C. psittaci; Helicobacter sp., e.g. H. pylori, etc.
[0150] Non bacterial pathogens of interest include fungal and
protozoan pathogens, e.g. Plasmodia sp., e.g. P. falciparum,
Trypanosoma sp., e.g. T. brucei; shistosomes; Entaemoeba sp.,
Cryptococcus sp., Candida sp., e.g. C. albicans; etc.
[0151] Various methods for administration may be employed. The
polypeptide formulation may be given orally, or may be injected
intravascularly, subcutaneously, peritoneally, by aerosol,
opthalmically, intra-bladder, topically, etc. For example, methods
of administration by inhalation are well-known in the art. The
dosage of the therapeutic formulation will vary widely, depending
on the specific antimicrobial polypeptide to be administered, the
nature of the disease, the frequency of administration, the manner
of administration, the clearance of the agent from the host, and
the like. The initial dose may be larger, followed by smaller
maintenance doses. The dose may be administered as infrequently as
weekly or biweekly, or fractionated into smaller doses and
administered once or several times daily, semi-weekly, etc. to
maintain an effective dosage level. In many cases, oral
administration will require a higher dose than if administered
intravenously. The amide bonds, as well as the amino and carboxy
termini, may be modified for greater stability on oral
administration. For example, the carboxy terminus may be
amidated.
Formulations
[0152] The compounds of this invention can be incorporated into a
variety of formulations for therapeutic administration. More
particularly, the compounds of the present invention can be
formulated into pharmaceutical compositions by combination with
appropriate, pharmaceutically acceptable carriers or diluents, and
may be formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, creams, foams, solutions, suppositories, injections,
inhalants, gels, microspheres, lotions, and aerosols. As such,
administration of the compounds can be achieved in various ways,
including oral, buccal, rectal, parenteral, intraperitoneal,
intradermal, transdermal, intracheal, etc., administration. The
antimicrobial polypeptides of the invention may be systemic after
administration or may be localized by the use of an implant or
other formulation that acts to retain the active dose at the site
of implantation.
[0153] In one embodiment, a formulation for topical use comprises a
chelating agent that decreases the effective concentration of
divalent cations, particularly calcium and magnesium. For example,
agents such as citrate, EGTA or EDTA may be included, where citrate
is preferred. The concentration of citrate will usually be from
about 1 to 10 mM.
[0154] The compounds of the present invention can be administered
alone, in combination with each other, or they can be used in
combination with other known compounds (e.g., perforin,
anti-inflammatory agents, antibiotics, etc.) In pharmaceutical
dosage forms, the compounds may be administered in the form of
their pharmaceutically acceptable salts. The following methods and
excipients are merely exemplary and are in no way limiting.
[0155] For oral preparations, the compounds can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0156] The compounds can be formulated into preparations for
injections by dissolving, suspending or emulsifying them in an
aqueous or nonaqueous solvent, such as vegetable or other similar
oils, synthetic aliphatic acid glycerides, esters of higher
aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0157] The compounds can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0158] The compounds can be used as lotions, for example to prevent
infection of burns, by formulation with conventional additives such
as solubilizers, isotonic agents, suspending agents, emulsifying
agents, stabilizers and preservatives.
[0159] Furthermore, the compounds can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0160] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more compounds of the present invention.
Similarly, unit dosage forms for injection or intravenous
administration may comprise the compound of the present invention
in a composition as a solution in sterile water, normal saline or
another pharmaceutically acceptable carrier.
[0161] Implants for sustained release formulations are well-known
in the art. Implants are formulated as microspheres, slabs, etc.
with biodegradable or non-biodegradable polymers. For example,
polymers of lactic acid and/or glycolic acid form an erodible
polymer that is well-tolerated by the host. The implant containing
the antimicrobial polypeptides of the invention is placed in
proximity to the site of infection, so that the local concentration
of active agent is increased relative to the rest of the body.
[0162] The term "unit dosage form", as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the unit dosage forms of the present invention
depend on the particular compound employed and the effect to be
achieved, and the pharmacodynamics associated with the compound in
the host.
[0163] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0164] Typical dosages for systemic administration range from 0.1
pg to 100 milligrams per kg weight of subject per administration. A
typical dosage may be one tablet taken from two to six times daily,
or one time-release capsule or tablet taken once a day and
containing a proportionally higher content of active ingredient.
The time-release effect may be obtained by capsule materials that
dissolve at different pH values, by capsules that release slowly by
osmotic pressure, or by any other known means of controlled
release.
[0165] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Some of the specific compounds are more potent than others.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means. A preferred means
is to measure the physiological potency of a given compound.
[0166] The use of liposomes as a delivery vehicle is one method of
interest. The liposomes fuse with the cells of the target site and
deliver the contents of the lumen intracellularly. The liposomes
are maintained in contact with the cells for sufficient time for
fusion, using various means to maintain contact, such as isolation,
binding agents, and the like. In one aspect of the invention,
liposomes are designed to be aerosolized for pulmonary
administration. Liposomes may be prepared with purified proteins or
peptides that mediate fusion of membranes, such as Sendai virus or
influenza virus, etc. The lipids may be any useful combination of
known liposome forming lipids, including cationic or zwitterionic
lipids, such as phosphatidylcholine. The remaining lipid will be
normally be neutral or acidic lipids, such as cholesterol,
phosphatidyl serine, phosphatidyl glycerol, and the like.
[0167] For preparing the liposomes, the procedure described by Kato
et al. (1991) J. Biol. Chem. 266:3361 may be used. Briefly, the
lipids and lumen composition containing peptides are combined in an
appropriate aqueous medium, conveniently a saline medium where the
total solids will be in the range of about 1-10 weight percent.
After intense agitation for short periods of time, from about 5-60
sec., the tube is placed in a warm water bath, from about
25-40.degree. C. and this cycle repeated from about 5-10 times. The
composition is then sonicated for a convenient period of time,
generally from about 1-10 sec. and may be further agitated by
vortexing. The volume is then expanded by adding aqueous medium,
generally increasing the volume by about from 1-2 fold, followed by
shaking and cooling. This method allows for the incorporation into
the lumen of high molecular weight molecules.
Formulations with Other Active Agents
[0168] For use in the subject methods, the antimicrobial
polypeptides of the invention may be formulated with other
pharmaceutically active agents, particularly other antimicrobial
agents. Other agents of interest include a wide variety of
antibiotics, as known in the art. Classes of antibiotics include
penicillins, e.g. penicillin G, penicillin V, methicillin,
oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins
in combination with beta-lactamase inhibitors, cephalosporins, e.g.
cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems;
monobactams; aminoglycosides; tetracyclines; macrolides;
lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical;
metronidazole; spectinomycin; trimethoprim; vancomycin; etc.
[0169] Anti-mycotic agents are also useful, Including polyenes,
e.g. amphotericin B, nystatin; 5-flucosyn; and azoles, e.g.
miconazol, ketoconazol, itraconazol and fluconazol.
Antituberculotic drugs include isoniazid, ethambutol, streptomycin
and rifampin. Cytokines may also be included in a formulation of
the antimicrobial polypeptides of the invention, e.g. interferon
gamma, tumor necrosis factor alpha, interleukin 12, etc.
In Vitro Synthesis
[0170] The antimicrobial peptides of the invention may be prepared
by in vitro synthesis, using conventional methods as known in the
art. Various commercial synthetic apparatuses are available, for
example automated synthesizers by Applied Biosystems Inc., Beckman,
etc. By using synthesizers, naturally occurring amino acids may be
substituted with unnatural amino acids, particularly D-isomers (or
D-forms) e.g. D-alanine and D-isoleucine, diastereoisomers, side
chains having different lengths or functionalities, and the like.
The particular sequence and the manner of preparation will be
determined by convenience, economics, purity required, and the
like.
[0171] Chemical linking may be provided to various peptides or
proteins comprising convenient functionalities for bonding, such as
amino groups for amide or substituted amine formation, e.g.
reductive amination, thiol groups for thioether or disulfide
formation, carboxyl groups for amide formation, and the like.
[0172] If desired, various groups may be introduced into the
peptide during synthesis or during expression, which allow for
linking to other molecules or to a surface. Thus cysteines can be
used to make thioethers, histidines for linking to a metal ion
complex, carboxyl groups for forming amides or esters, amino groups
for forming amides, and the like.
[0173] The polypeptides may also be isolated and purified in
accordance with conventional methods of recombinant synthesis. A
lysate may be prepared of the expression host and the lysate
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, or other purification technique. For the
most part, the compositions which are used will comprise at least
20% by weight of the desired product, more usually at least about
75% by weight, preferably at least about 95% by weight, and for
therapeutic purposes, usually at least about 99.5% by weight, in
relation to contaminants related to the method of preparation of
the product and its purification. Usually, the percentages will be
based upon total protein
Animal Feed
[0174] The present invention is also directed to methods for using
the polypeptides having antimicrobial activity in animal feed, as
well as to feed compositions and feed additives comprising the
antimicrobial polypeptides of the invention.
[0175] The term animal includes all animals, including human
beings. Examples of animals are non-ruminants, and ruminants, such
as cows, sheep and horses. In a particular embodiment, the animal
is a non-ruminant animal. Non-ruminant animals include mono-gastric
animals, e.g. pigs or swine (including, but not limited to,
piglets, growing pigs, and sows); poultry such as turkeys and
chicken (including but not limited to broiler chicks, layers);
young calves; and fish (including but not limited to salmon).
[0176] The term feed or feed composition means any compound,
preparation, mixture, or composition suitable for, or intended for
intake by an animal.
[0177] In the use according to the invention the antimicrobial
polypeptide can be fed to the animal before, after, or
simultaneously with the diet. The latter is preferred.
[0178] In a particular embodiment, the antimicrobial polypeptide,
in the form in which it is added to the feed, or when being
included in a feed additive, is well defined. Well-defined means
that the antimicrobial polypeptide preparation is at least 50% pure
as determined by Size-exclusion chromatography (see Example 12 of
WO 01/58275). In other particular embodiments the antimicrobial
polypeptide preparation is at least 60, 70, 80, 85, 88, 90, 92, 94,
or at least 95% pure as determined by this method.
[0179] A well-defined antimicrobial polypeptide preparation is
advantageous. For instance, it is much easier to dose correctly to
the feed an antimicrobial polypeptide that is essentially free from
interfering or contaminating other antimicrobial polypeptides. The
term dose correctly refers in particular to the objective of
obtaining consistent and constant results, and the capability of
optimising dosage based upon the desired effect.
[0180] For the use in animal feed, however, the antimicrobial
polypeptide need not be that pure; it may e.g. include other
enzymes, in which case it could be termed an antimicrobial
polypeptide preparation.
[0181] The antimicrobial polypeptide preparation can be (a) added
directly to the feed (or used directly in a treatment process of
vegetable proteins), or (b) it can be used in the production of one
or more intermediate compositions such as feed additives or
premixes that is subsequently added to the feed (or used in a
treatment process). The degree of purity described above refers to
the purity of the original antimicrobial polypeptide preparation,
whether used according to (a) or (b) above.
[0182] Antimicrobial polypeptide preparations with purities of this
order of magnitude are in particular obtainable using recombinant
methods of production, whereas they are not so easily obtained and
also subject to a much higher batch-to-batch variation when the
antimicrobial polypeptide is produced by traditional fermentation
methods.
[0183] Such antimicrobial polypeptide preparation may of course be
mixed with other enzymes.
[0184] The term vegetable proteins as used herein refers to any
compound, composition, preparation or mixture that includes at
least one protein derived from or originating from a vegetable,
including modified proteins and protein-derivatives. In particular
embodiments, the protein content of the vegetable proteins is at
least 10, 20, 30, 40, 50, or 60% (w/w).
[0185] Vegetable proteins may be derived from vegetable protein
sources, such as legumes and cereals, for example materials from
plants of the families Fabaceae (Leguminosae), Cruciferaceae,
Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal and
rapeseed meal.
[0186] In a particular embodiment, the vegetable protein source is
material from one or more plants of the family Fabaceae, e.g.
soybean, lupine, pea, or bean.
[0187] In another particular embodiment, the vegetable protein
source is material from one or more plants of the family
Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa.
[0188] Other examples of vegetable protein sources are rapeseed,
and cabbage.
[0189] Soybean is a preferred vegetable protein source.
[0190] Other examples of vegetable protein sources are cereals such
as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
[0191] The antimicrobial polypeptide can be added to the feed in
any form, be it as a relatively pure antimicrobial polypeptide, or
in admixture with other components intended for addition to animal
feed, i.e. in the form of animal feed additives, such as the
so-called pre-mixes for animal feed.
[0192] In a further aspect the present invention relates to
compositions for use in animal feed, such as animal feed, and
animal feed additives, e.g. premixes.
[0193] Apart from the antimicrobial polypeptide of the invention,
the animal feed additives of the invention contain at least one fat
soluble vitamin, and/or at least one water soluble vitamin, and/or
at least one trace mineral, and/or at least one macro mineral.
[0194] Further, optional, feed-additive ingredients are colouring
agents, aroma compounds, stabilisers, and/or at least one other
enzyme selected from amongst phytases EC 3.1.3.8 or 3.1.3.26;
xylanases EC 3.2.1.8; galactanases EC 3.2.1.89; and/or
beta-glucanases EC 3.2.1.4.
[0195] In a particular embodiment these other enzymes are well
defined (as defined above for antimicrobial polypeptide
preparations).
[0196] Examples of other antimicrobial peptides (AMP's) are CAP18,
Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensin, Ovispirin
such as Novispirin (Robert Lehrer, 2000), and variants, or
fragments thereof which retain antimicrobial activity.
[0197] Examples of other antifungal polypeptides (AFP's) are the
Aspergillus giganteus, and Aspergillus niger peptides, as well as
variants and fragments thereof which retain antifungal activity, as
disclosed in WO 94/01459 and PCT/DK02/00289 [replace with WO number
once published].
[0198] Usually fat and water soluble vitamins, as well as trace
minerals form part of a so-called premix intended for addition to
the feed, whereas macro minerals are usually separately added to
the feed. Either of these composition types, when enriched with an
antimicrobial polypeptide of the invention, is an animal feed
additive of the invention.
[0199] In a particular embodiment, the animal feed additive of the
invention is intended for being included (or prescribed as having
to be included) in animal diets or feed at levels of 0.01 to 10.0%;
more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g
additive per 100 g feed). This is so in particular for
premixes.
[0200] The following are non-exclusive lists of examples of these
components:
[0201] Examples of fat soluble vitamins are vitamin A, vitamin D3,
vitamin E, and vitamin K, e.g. vitamin K3.
[0202] Examples of water soluble vitamins are vitamin B12, biotin
and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid
and panthothenate, e.g. Ca-D-panthothenate.
[0203] Examples of trace minerals are manganese, zinc, iron,
copper, iodine, selenium, and cobalt.
[0204] Examples of macro minerals are calcium, phosphorus and
sodium. The nutritional requirements of these components
(exemplified with poultry and piglets/pigs) are listed in Table A
of WO 01/58275. Nutritional requirement means that these components
should be provided in the diet in the concentrations indicated.
[0205] In the alternative, the animal feed additive of the
invention comprises at least one of the individual components
specified in Table A of WO 01/58275. At least one means either of,
one or more of, one, or two, or three, or four and so forth up to
all thirteen, or up to all fifteen individual components. More
specifically, this at least one individual component is included in
the additive of the invention in such an amount as to provide an
in-feed-concentration within the range indicated in column four, or
column five, or column six of Table A.
[0206] The present invention also relates to animal feed
compositions. Animal feed compositions or diets have a relatively
high content of protein. Poultry and pig diets can be characterised
as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can
be characterised as indicated in column 4 of this Table B.
Furthermore such fish diets usually have a crude fat content of
200-310 g/kg.
[0207] An animal feed composition according to the invention has a
crude protein content of 50-800 g/kg, and furthermore comprises at
least one antimicrobial polypeptide as claimed herein.
[0208] Furthermore, or in the alternative (to the crude protein
content indicated above), the animal feed composition of the
invention has a content of metabolizable energy of 10-30 MJ/kg;
and/or a content of calcium of 0.1-200 g/kg; and/or a content of
available phosphorus of 0.1-200 g/kg; and/or a content of
methionine of 0.1-100 g/kg; and/or a content of methionine plus
cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50
g/kg.
[0209] In particular embodiments, the content of metabolizable
energy, crude protein, calcium, phosphorus, methionine, methionine
plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or
5 in Table B of WO 01/58275 (R. 2-5).
[0210] Crude protein is calculated as nitrogen (N) multiplied by a
factor 6.25, i.e. Crude protein (g/kg)=N (g/kg).times.6.25. The
nitrogen content is determined by the Kjeldahl method (A.O.A.C.,
1984, Official Methods of Analysis 14th ed., Association of
Official Analytical Chemists, Washington D.C.).
[0211] Metabolizable energy can be calculated on the basis of the
NRC publication Nutrient requirements in swine, ninth revised
edition 1988, subcommittee on swine nutrition, committee on animal
nutrition, board of agriculture, national research council.
National Academy Press, Washington, D.C., pp. 2-6, and the European
Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre
for poultry research and extension, 7361 DA Beekbergen, The
Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen.
ISBN 90-71463-12-5.
[0212] The dietary content of calcium, available phosphorus and
amino acids in complete animal diets is calculated on the basis of
feed tables such as Veevoedertabel 1997, gegevens over chemische
samenstelling, verteerbaarheid en voederwaarde van voedermiddelen,
Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN
90-72839-13-7.
[0213] In a particular embodiment, the animal feed composition of
the invention contains at least one vegetable protein or protein
source as defined above.
[0214] In still further particular embodiments, the animal feed
composition of the invention contains 0-80% maize; and/or 0-80%
sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30%
oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or
0-20% whey. Animal diets can e.g. be manufactured as mash feed (non
pelleted) or pelleted feed. Typically, the milled feed-stuffs are
mixed and sufficient amounts of essential vitamins and minerals are
added according to the specifications for the species in question.
Enzymes can be added as solid or liquid enzyme formulations. For
example, a solid enzyme formulation is typically added before or
during the mixing step; and a liquid enzyme preparation is
typically added after the pelleting step. The enzyme may also be
incorporated in a feed additive or premix.
[0215] The final enzyme concentration in the diet is within the
range of 0.01-200 mg enzyme protein per kg diet, for example in the
range of 5-30 mg enzyme protein per kg animal diet.
[0216] The antimicrobial polypeptide may be administered in one or
more of the following amounts (dosage ranges): 0.01-200; or
0.01-100; or 0.05-100; or 0.05-50; or 0.10-10--all these ranges
being in mg antimicrobial polypeptide protein per kg feed
(ppm).
[0217] For determining mg antimicrobial polypeptide protein per kg
feed, the antimicrobial polypeptide is purified from the feed
composition, and the specific activity of the purified
antimicrobial polypeptide is determined using a relevant assay (see
under antimicrobial activity, substrates, and assays). The
antimicrobial activity of the feed composition as such is also
determined using the same assay, and on the basis of these two
determinations, the dosage in mg antimicrobial polypeptide protein
per kg feed is calculated.
[0218] The same principles apply for determining mg antimicrobial
polypeptide protein in feed additives. Of course, if a sample is
available of the antimicrobial polypeptide used for preparing the
feed additive or the feed, the specific activity is determined from
this sample (no need to purify the antimicrobial polypeptide from
the feed composition or the additive).
Detergent Composition
[0219] The antimicrobial polypeptides of the invention may be added
to and thus become a component of a detergent composition.
[0220] The detergent composition of the invention may for example
be formulated as a hand or machine laundry detergent composition
including a laundry additive composition suitable for pre-treatment
of stained fabrics and a rinse added fabric softener composition,
or be formulated as a detergent composition for use in general
household hard surface cleaning operations, or be formulated for
hand or machine dishwashing operations.
[0221] In a specific aspect, the invention provides a detergent
additive comprising the antimicrobial polypeptides of the invention
and a surfactant. The detergent additive as well as the detergent
composition may comprise one or more other enzymes such as a
protease, a lipase, a cutinase, an amylase, a carbohydrase, a
cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a
xylanase, an oxidase (such as a laccase), and/or a peroxidase (such
as a haloperoxidase).
[0222] In general the properties of the chosen enzyme(s) should be
compatible with the selected detergent, (i.e. pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective
amounts.
[0223] Proteases: Suitable proteases include those of animal,
vegetable or microbial origin. Microbial origin is preferred.
Chemically modified or protein engineered mutants are included. The
protease may be a serine protease or a metallo protease, preferably
an alkaline microbial protease or a trypsin-like protease. Examples
of alkaline proteases are subtilisins, especially those derived
from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg,
subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO
89/06279). Examples of trypsin-like proteases are trypsin (e.g. of
porcine or bovine origin) and the Fusarium protease described in WO
89/06270 and WO 94/25583.
[0224] Examples of useful proteases are the variants described in
WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially
the variants with substitutions in one or more of the following
positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170,
194, 206, 218, 222, 224, 235 and 274.
[0225] Lipases: Suitable lipases include those of bacterial or
fungal origin. Chemically modified or protein engineered mutants
are included. Examples of useful lipases include lipases from
Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T.
lanuginosus) as described in EP 258 068 and EP 305 216 or from H.
insolens as described in WO 96/13580, a Pseudomonas lipase, e.g.
from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.
cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,
Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.
wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B.
subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,
1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus
(WO 91/16422).
[0226] Other examples are lipase variants such as those described
in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381,
WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO
97/04079 and WO 97/07202.
[0227] Amylases: Suitable amylases (alpha and/or beta) include
those of bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Amylases include, for example,
alpha-amylases obtained from Bacillus, e.g. a special strain of B.
licheniformis, described in more detail in GB 1,296,839.
[0228] Examples of useful amylases are the variants described in WO
94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the
variants with substitutions in one or more of the following
positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188,
190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.
[0229] Cellulases: Suitable cellulases include those of bacterial
or fungal origin. Chemically modified or protein engineered mutants
are included. Suitable cellulases include cellulases from the
genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,
Acremonium, e.g. the fungal cellulases produced from Humicola
insolens, Myceliophthora thermophila and Fusarium oxysporum
disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S.
Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.
[0230] Especially suitable cellulases are the alkaline or neutral
cellulases having colour care benefits. Examples of such cellulases
are cellulases described in EP 0 495 257, EP 0 531 372, WO
96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase
variants such as those described in WO 94/07998, EP 0 531 315, U.S.
Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No.
5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
[0231] Peroxidases/Oxidases: Suitable peroxidases/oxidases include
those of plant, bacterial or fungal origin. Chemically modified or
protein engineered mutants are included. Examples of useful
peroxidases include peroxidases from Coprinus, e.g. from C.
cinereus, and variants thereof as those described in WO 93/24618,
WO 95/10602, and WO 98/15257.
[0232] The detergent enzyme(s) may be included In a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive of the invention, i.e. a separate
additive or a combined additive, can be formulated e.g. as a
granulate, a liquid, a slurry, etc. Preferred detergent additive
formulations are granulates, in particular non-dusting granulates,
liquids, in particular stabilized liquids, or slurries.
[0233] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (polyethyleneglycol,
PEG) with mean molar weights of 1000 to 20000; ethoxylated
nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated
fatty alcohols in which the alcohol contains from 12 to 20 carbon
atoms and in which there are 15 to 80 ethylene oxide units; fatty
alcohols; fatty acids; and mono- and di- and triglycerides of fatty
acids. Examples of film-forming coating materials suitable for
application by fluid bed techniques are given in GB 1483591. Liquid
enzyme preparations may, for instance, be stabilized by adding a
polyol such as propylene glycol, a sugar or sugar alcohol, lactic
acid or boric acid according to established methods. Protected
enzymes may be prepared according to the method disclosed in EP
238,216.
[0234] The detergent composition of the invention may be in any
convenient form, e.g., a bar, a tablet, a powder, a granule, a
paste or a liquid. A liquid detergent may be aqueous, typically
containing up to 70% water and O-30% organic solvent, or
non-aqueous.
[0235] The detergent composition comprises one or more surfactants,
which may be non-ionic including semi-polar and/or anionic and/or
cationic and/or zwitterionic. The surfactants are typically present
at a level of from 0.1% to 60% by weight.
[0236] When included therein the detergent will usually contain
from about 1% to about 40% of an anionic surfactant such as linear
alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty
alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid
or soap.
[0237] When included therein the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl N-alkyl derivatives of glucosamine ("glucamides").
[0238] The detergent may contain 0-65% of a detergent builder or
complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, carbonate, citrate, nitrilotriacetic acid,
ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic
acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered
silicates (e.g. SKS-6 from Hoechst).
[0239] The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene
glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates such as polyacrylates,
maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid
copolymers.
[0240] The detergent may contain a bleaching system which may
comprise a H2O2 source such as perborate or percarbonate which may
be combined with a peracid-forming bleach activator such as
tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
Alternatively, the bleaching system may comprise peroxyacids of
e.g. the amide, imide, or sulfone type.
[0241] The enzyme(s) of the detergent composition of the invention
may be stabilized using conventional stabilizing agents, e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, boric acid, or a boric acid derivative, e.g.,
an aromatic borate ester, or a phenyl boronic acid derivative such
as 4-formylphenyl boronic acid, and the composition may be
formulated as described in e.g. WO 92/19709 and WO 92/19708.
[0242] The detergent may also contain other conventional detergent
ingredients such as e.g. fabric conditioners including clays, foam
boosters, suds suppressors, anti-corrosion agents, soil-suspending
agents, anti-soil redeposition agents, dyes, bactericides, optical
brighteners, hydrotropes, tarnish inhibitors, or perfumes.
[0243] It is at present contemplated that in the detergent
compositions any enzyme, and the antimicrobial polypeptides of the
invention, may be added in an amount corresponding to 0.01-100 mg
of enzyme protein per liter of wash liquor, preferably 0.05-10 mg
of enzyme protein per liter of wash liquor, more preferably 0.1-5
mg of enzyme protein per liter of wash liquor, and most preferably
0.1-1 mg of enzyme protein per liter of wash liquor.
[0244] The antimicrobial polypeptides of the invention may
additionally be incorporated in the detergent formulations
disclosed in WO 97/07202 which is hereby incorporated as
reference.
[0245] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
EXAMPLES
[0246] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
Example 1
Cloning, Expression and Activity Evaluation of an Antimicrobial
Polypeptide (Gen2)
Cloning of Synthetic Gene Encoding Gen2
[0247] In order to produce the antimicrobial polypeptide Gen2 (SEQ
ID NO:2) for antimicrobial activity assays, a synthetic gene was
made (see below) and inserted into the expression vector
pET31b+(Novagen Inc.). The synthetic gene was constituted by
specifically designed oligonucleotides (Primer1 and Primer2).
TABLE-US-00001 Synthetic gene encoding Gen2 (SEQ ID NO:47): GGC CTG
CTG AGC AAA CTG AAG AAG GCG GCG AAA AAA G L L S K L K K A A K K GCG
CTG AAA CAT GTG CTG TAG A L K H V L * Primer1 (SEQ ID NO:49):
ATTATTCAGA TGCTGGATCC GGCGGAAGGC CTGCTGAGCA AACTGAAGAA GGCGGCGAAA
AAAGCGCTGA AACATGTGCT GTAGCTCGAG ATTATT Primer2 (SEQ ID NO:50):
AATAATCTCG AGCTACAGCA CATGTTTCAG CGCTTTTTTC GCCGCCTTCT TCAGTTTGCT
CAGCAGGCCT TCCGCCGGAT CCAGCATCTG AATAAT
[0248] Enzymatic digestion of flanking restriction endonuclease
sites (AlwNI/AvaI) enabled us to clone this synthetic gene as a
fusion construct in pET31 b+(standard procedures as described by
the manufacturer, New England Biolabs Inc.). All standard protocols
have been described elsewhere (Sambrook, Fritsch, and Maniatis,
1989).
Transformation and Expression of Gen2 in E. Coli
[0249] Recombinant pET31 b+was transformed into E. coli Novablue as
described by the manufacturer (Novagen). Plasmid was prepared by
QIAprep Mini Columns (QIAGEN Inc.) and sequenced by automated
sequencing using plasmid specific primers (Primer3 and Primer4):
TABLE-US-00002 Primer3 (SEQ ID NO:51): TGCTA GTTAT TGCTC AGCGG
Primer4 (SEQ ID NO:52): ACCGT AGTTG CGCCC ATCG
[0250] Plasmid was transformed in E. coli BLR-DE3 according to the
manufacturer (Novagen). Bacteria were cultivated in LB media to
OD.sub.600.about.0.8 and recombinant protein synthesis was
initiated by 1 mM IPTG (Isopropyl beta-D-Thiogalactopyranoside).
Upon 3 hours of induction, bacteria were harvested, re-suspended in
1/10 volume buffer A (50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8)
and lysed by pressure disruption (1500 mBar). Resulting pellet was
washed twice in buffer B (50 mM Tris-HCl, 10 mM EDTA, 0.5%
TritonX-100, 100 mM NaCl, pH 8). All standard protocols have been
described elsewhere (Sambrook, Fritsch, and Maniatis, 1989).
Purification of Gen2 from E. Coli Inclusion Bodies
[0251] The pellet resulting from the above purification contained
purified inclusion bodies. To liberate the peptide from the KSI
fusion partner, acid hydrolysis was performed on an engineered
Asp-Pro site, introduced N-terminally to the gene encoding Gen2.
Inclusion bodies were re-suspended in 100 mM sodium phosphate (pH
2.3) and incubated overnight at 85 degrees Celsius. Resulting
supernatant contained peptide (PAE-Gen2). The sample was
neutralized by adding 100 mM sodium phosphate (pH 12.3). In order
to maturate the peptide, the peptide was treated with a glutamyl
endopeptidase I (from B. licheniformis). The maturated peptide was
confirmed by mass-spectrometry and further purified by standard
chromatographic procedures.
Evaluation of Minimal Inhibitory Concentration (MIC)
[0252] Minimal inhibitory concentrations of Gen2 were determined
using cation-adjusted Mueller hinton broth (MHB) in the microbroth
dilution assay, wherein MHB was used with different modifications
as depicted in Table 1 below. The selected test organism was
Pseudomonas aeroginosa (ATCC 27853). TABLE-US-00003 TABLE 1 Test
conditions MIC (.mu.g/ml) MHB 15 MHB + 1.5 mM Mg.sup.2+ 40 MHB +
Human Serum Albumin (1 mg/ml) 36 MHB + 1% Human Serum 28 MHB + 1%
lysed human red blood cells 36 MHB adjusted to pH 6.5 16 MHB
adjusted to pH 5.5 16
Killing Kinetics
[0253] The rate of killing of Gen2 was determined in MHB using
Pseudomonas aeroginosa (ATCC 27853) as the test organism. The
bacteria were incubated with Gen2 (at 4.times.MIC concentration).
At different time-points, several dilutions of the preparation were
plated and an estimate of viable cells was determined (CFU/ml). The
results in Table 2 indicate that Gen2 rapidly kills the bacteria,
with a near 3-log reduction in CFU within 30 minutes.
TABLE-US-00004 TABLE 2 CFU/ml Time Control Gen2 0 hours 100.000
100.000 0.5 hours 100.000 115 1.5 hours 300.000 60 24 hours
>1.000.000 <20
Antimicrobial Activity by Radial Diffusion Assay (MEC)
[0254] A modified version of a previously published protocol has
been applied in the detection of antimicrobial activity (Lehrer et
al., (1991) Ultra sensitive assays for endogenous antimicrobial
polypeptides J Immunol Methods 137: 167-173). Target bacteria
(10.sup.6 colony forming units (CFU)) were added to 10 ml of
underlay agarose (1% low electro-endosmosis agarose, 0.03%
Trypticase soy broth, 10 mM sodium phosphate, pH 7.4, 37 degrees
Celsius). Suspension was solidified on an INTEGRID Petri Dish
(Becton Dickinson Labware). A 3 mm Gel Puncher was used to make
holes in the underlay agarose (Amersham Pharmacia Biotech, Sweden).
Samples were added to the holes and incubated at 37 degrees
Celsius, 3 hours. An overlay was poured on top and the plate was
incubated overnight (LB media, 7.5% Agar). Antimicrobial activity
was seen as bacterial clearing zones around the wells. Living cells
was counterstained by adding 10 ml, 0.2 mM MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Thiazolyl blue). This assay revealed antimicrobial activity (<64
.mu.g/ml) of Gen2 against both Bacillus subtilis & Eschericia
coli.
Example 2
Evaluation of Antimicrobial Activity
[0255] A range of synthetic antimicrobial polypeptides were
expressed in E. coli TOP10 (Invitrogen) using arabinose as inducer,
as disclosed in Example 1 of International patent application WO
00/73433 (with minor modifications). Expression of the
antimicrobial polypeptides of the invention resulted in growth
inhibition of the host cells.
[0256] Briefly, fresh overnight cultures grown in RM medium
containing 0.2% glucose and ampicillin (100 .mu.g/ml) were diluted
300-fold into 150 microliter of RM containing 0.2% glycerol and
ampicillin (100 .mu.g/ml) and different concentrations of arabinose
(0, 0.01% or 0.1%) in a microtiter plate and incubated at 37
degrees Celsius with vigorous shaking. The growth curve was
monitored by measuring OD450 using a Bioscreen C Microbiology
reader (Thermo Electron Corporation) at intervals of 30 minutes for
14 hours (see Invitrogen protocol of the pBAD/gill A, B and C
vectors catalogue nos. V450-01 for buffer and media
composition).
[0257] The percentage of growth inhibition was calculated as the
end point OD measurement of each sample divided by the end point OD
measurement obtained from cells containing the control vector and
multiplied by 100. The formula is the following: ( 1 - sample
.times. .times. OD - blank .times. .times. OD control .times.
.times. vector .times. .times. OD - blank .times. .times. OD ) 100
##EQU1## where "blank OD" corresponds to the OD of an empty
well.
[0258] The amino acid sequences of Gen2 and selected improved
synthetic variants are listed in Table 3. TABLE-US-00005 TABLE 3
Growth Growth SEQ inhibition inhibition ID using 0.01% using 0.1%
Amino acid sequence NO: arabinose (%) arabinose (%)
GLLSKLKKAAKKALKHVL 2 51 48 (Gen2) GLFTKLRKATKRILEHVL 3 81 87
GLLKTIRKKIKRVLKHVR 4 96 99 GLWRKLKKALKRAVQGVR 5 91 96
GMLSKLGITIKIAVKHIR 6 98 100 GWFSKLKKTAKKLLQRVL 7 76 82
GMLRKLKRKVKRTLQHVL 8 78 88 GLFSIIMRAVKKVWQRVR 9 97 100
GLLRKLGKKIKRVVKHVG 10 98 100 GWFNKLKTKIKKTLKHVL 11 72 90
GLLEKLRKALKRILQHVL 12 91 97 GLWRKLRRKAKKVLQHIL 13 94 100
GLLSRLRRATKIILKGIR 14 72 88 GLWNMLKKKLKKIAQGIR 15 73 88
GLLSKIMKAVKRTLKHIL 16 89 99 GMLIKLEMEAKKVVKNVL 17 96 99
GLLNKIKKTIKRAVQHVL 18 76 92 GLLSKLKKTVKRVVKHVR 19 84 99
GLLSKIRKKLKRVLQSIR 20 77 94 GLVTLLKKAMKNALEDVL 21 98 99
GLLRKIKMKAKKVLKNIL 22 72 83 GLFRKLRKKVKKVLKHVL 23 91 98
GLLSILKRKSKRILKHIL 24 88 96 GILNIIGRAVKTVLESIR 25 97 99
GLLSMLGKAVKRAVQHVL 26 97 100 GILNKLRKKLKRVLQRIL 27 84 97
GLLSKLGKAVKNILEDVV 28 98 100 GLWSSIKKEAKHALKHIL 29 55 81
GLLSKLKRKIKKAVKHIL 30 82 95 GLFRKLKKTIKRVLKHVP 31 86 96
GLFSLLRKTIKKVLQHIR 32 91 99 GLLNKLKRALKKVVKHVR 33 84 94
GWLRKIGKAVKKVVKRVL 34 90 99 GLLGKLKRKIKKALEGIR 35 96 99
GWLKILEKAAKITVKNVL 36 98 97 GLLRILKKKAKKALQHIL 37 86 93
GLLGKIRKEGRNFWRVFRRWL 38 86 92 GLLRKLRKEVKKVLSIFFRWL 39 97 99
GLLNKLKKNVKNIVQHILRWL 40 77 95 GLFSILKREIKRTFSNFYRWL 41 86 99
GLLGKLKKELKNVLEHIYRWL 42 75 93 GILSKLKKAIKRILQDVLRWL 43 82 99
GMLKKLKKKTKRAFSVFCRWL 44 96 98 GILSMLERRWSMYCSIFCRWL 45 98 100
GILSKLKKKAKNAVKMFCRWL 46 92 96
[0259] The results shown in Table 3 indicate convincingly that all
the tested antimicrobial polypeptides exhibit strong antimicrobial
activity.
Sequence CWU 1
1
52 1 21 PRT Artificial Synthetic antimicrobial polypeptide
MISC_FEATURE (2)..(2) Xaa = leucine, isoleucine, tryptophan or
methionine MISC_FEATURE (3)..(3) Xaa = leucine, phenylalanine,
tryptophan or valine MISC_FEATURE (4)..(4) Xaa = serine, glycine,
lysine, threonine, arginine, isoleucine,asparagine, aspartic acid
or glutamic acid MISC_FEATURE (5)..(5) Xaa = lysine, threonine,
phenylalanine, arginine, leucine, isoleucine, methionine or serine
MISC_FEATURE (6)..(6) Xaa = leucine or isoleucine MISC_FEATURE
(7)..(7) Xaa = lysine, glycine, arginine, methionine or glutamic
acid MISC_FEATURE (8)..(8) Xaa = lysine, serine, isoleucine,
arginine, threonine or methionine MISC_FEATURE (9)..(9) Xaa =
alanine, lysine, threonine, asparagine, arginine or glutamic acid
MISC_FEATURE (10)..(10) Xaa = alanine, glycine, serine, isoleucine,
leucine, threonine, valine, methionine or tryptophan MISC_FEATURE
(11)..(11) Xaa = serine, arginine, lysine or glutamic acid
MISC_FEATURE (12)..(12) Xaa = lysine, methionine, arginine,
histidine, isoleucine, asparagine or threonine MISC_FEATURE
(13)..(13) Xaa = alanine, valine, isoleucine, leucine,
tyrosine,phenylalanine or threonine MISC_FEATURE (14)..(14) Xaa =
leucine, alanine, glycine, cysteine, phenylalanine, valineor
tryptophan MISC_FEATURE (15)..(15) Xaa = lysine, glutamine,
alanine, serine, arginine or glutamic acid MISC_FEATURE (16)..(16)
Xaa = histidine, glycine, asparagine, arginine, serine,methionine,
isoleucine, valine or aspartic acid MISC_FEATURE (17)..(17) Xaa =
valine, isoleucine, alanine or phenylalanine MISC_FEATURE
(18)..(18) Xaa = phenylalanine, leucine, arginine, alanine,
glycine, valine,tyrosine, cysteine or proline MISC_FEATURE
(19)..(19) Xaa = arginine or is absent MISC_FEATURE (20)..(20) Xaa
= tryptophan or is absent MISC_FEATURE (21)..(21) Xaa = leucine or
is absent 1 Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 2 18 PRT Artificial
Synthetic antimicrobial peptide 2 Gly Leu Leu Ser Lys Leu Lys Lys
Ala Ala Lys Lys Ala Leu Lys His 1 5 10 15 Val Leu 3 18 PRT
Artificial Synthetic antimicrobial peptide 3 Gly Leu Phe Thr Lys
Leu Arg Lys Ala Thr Lys Arg Ile Leu Glu His 1 5 10 15 Val Leu 4 18
PRT Artificial Synthetic antimicrobial peptide 4 Gly Leu Leu Lys
Thr Ile Arg Lys Lys Ile Lys Arg Val Leu Lys His 1 5 10 15 Val Arg 5
18 PRT Artificial Synthetic antimicrobial peptide 5 Gly Leu Trp Arg
Lys Leu Lys Lys Ala Leu Lys Arg Ala Val Gln Gly 1 5 10 15 Val Arg 6
18 PRT Artificial Synthetic antimicrobial peptide 6 Gly Met Leu Ser
Lys Leu Gly Ile Thr Ile Lys Ile Ala Val Lys His 1 5 10 15 Ile Arg 7
18 PRT Artificial Synthetic antimicrobial peptide 7 Gly Trp Phe Ser
Lys Leu Lys Lys Thr Ala Lys Lys Leu Leu Gln Arg 1 5 10 15 Val Leu 8
18 PRT Artificial Synthetic antimicrobial peptide 8 Gly Met Leu Arg
Lys Leu Lys Arg Lys Val Lys Arg Thr Leu Gln His 1 5 10 15 Val Leu 9
18 PRT Artificial Synthetic antimicrobial peptide 9 Gly Leu Phe Ser
Ile Ile Met Arg Ala Val Lys Lys Val Trp Gln Arg 1 5 10 15 Val Arg
10 18 PRT Artificial Synthetic antimicrobial peptide 10 Gly Leu Leu
Arg Lys Leu Gly Lys Lys Ile Lys Arg Val Val Lys His 1 5 10 15 Val
Gly 11 18 PRT Artificial Synthetic antimicrobial peptide 11 Gly Trp
Phe Asn Lys Leu Lys Thr Lys Ile Lys Lys Thr Leu Lys His 1 5 10 15
Val Leu 12 18 PRT Artificial Synthetic antimicrobial peptide 12 Gly
Leu Leu Glu Lys Leu Arg Lys Ala Leu Lys Arg Ile Leu Gln His 1 5 10
15 Val Leu 13 18 PRT Artificial Synthetic antimicrobial peptide 13
Gly Leu Trp Arg Lys Leu Arg Arg Lys Ala Lys Lys Val Leu Gln His 1 5
10 15 Ile Leu 14 18 PRT Artificial Synthetic antimicrobial peptide
14 Gly Leu Leu Ser Arg Leu Arg Arg Ala Thr Lys Ile Ile Leu Lys Gly
1 5 10 15 Ile Arg 15 18 PRT Artificial Synthetic antimicrobial
peptide 15 Gly Leu Trp Asn Met Leu Lys Lys Lys Leu Lys Lys Ile Ala
Gln Gly 1 5 10 15 Ile Arg 16 18 PRT Artificial Synthetic
antimicrobial peptide 16 Gly Leu Leu Ser Lys Ile Met Lys Ala Val
Lys Arg Thr Leu Lys His 1 5 10 15 Ile Leu 17 18 PRT Artificial
Synthetic antimicrobial peptide 17 Gly Met Leu Ile Lys Leu Glu Met
Glu Ala Lys Lys Val Val Lys Asn 1 5 10 15 Val Leu 18 18 PRT
Artificial Synthetic antimicrobial peptide 18 Gly Leu Leu Asn Lys
Ile Lys Lys Thr Ile Lys Arg Ala Val Gln His 1 5 10 15 Val Leu 19 18
PRT Artificial Synthetic antimicrobial peptide 19 Gly Leu Leu Ser
Lys Leu Lys Lys Thr Val Lys Arg Val Val Lys His 1 5 10 15 Val Arg
20 18 PRT Artificial Synthetic antimicrobial peptide 20 Gly Leu Leu
Ser Lys Ile Arg Lys Lys Leu Lys Arg Val Leu Gln Ser 1 5 10 15 Ile
Arg 21 18 PRT Artificial Synthetic antimicrobial peptide 21 Gly Leu
Val Thr Leu Leu Lys Lys Ala Met Lys Asn Ala Leu Glu Asp 1 5 10 15
Val Leu 22 18 PRT Artificial Synthetic antimicrobial peptide 22 Gly
Leu Leu Arg Lys Ile Lys Met Lys Ala Lys Lys Val Leu Lys Asn 1 5 10
15 Ile Leu 23 18 PRT Artificial Synthetic antimicrobial peptide 23
Gly Leu Phe Arg Lys Leu Arg Lys Lys Val Lys Lys Val Leu Lys His 1 5
10 15 Val Leu 24 18 PRT Artificial Synthetic antimicrobial peptide
24 Gly Leu Leu Ser Ile Leu Lys Arg Lys Ser Lys Arg Ile Leu Lys His
1 5 10 15 Ile Leu 25 18 PRT Artificial Synthetic antimicrobial
peptide 25 Gly Ile Leu Asn Ile Ile Gly Arg Ala Val Lys Thr Val Leu
Glu Ser 1 5 10 15 Ile Arg 26 18 PRT Artificial Synthetic
antimicrobial peptide 26 Gly Leu Leu Ser Met Leu Gly Lys Ala Val
Lys Arg Ala Val Gln His 1 5 10 15 Val Leu 27 18 PRT Artificial
Synthetic antimicrobial peptide 27 Gly Ile Leu Asn Lys Leu Arg Lys
Lys Leu Lys Arg Val Leu Gln Arg 1 5 10 15 Ile Leu 28 18 PRT
Artificial Synthetic antimicrobial peptide 28 Gly Leu Leu Ser Lys
Leu Gly Lys Ala Val Lys Asn Ile Leu Glu Asp 1 5 10 15 Val Val 29 18
PRT Artificial Synthetic antimicrobial peptide 29 Gly Leu Trp Ser
Ser Ile Lys Lys Glu Ala Lys His Ala Leu Lys His 1 5 10 15 Ile Leu
30 18 PRT Artificial Synthetic antimicrobial peptide 30 Gly Leu Leu
Ser Lys Leu Lys Arg Lys Ile Lys Lys Ala Val Lys His 1 5 10 15 Ile
Leu 31 18 PRT Artificial Synthetic antimicrobial peptide 31 Gly Leu
Phe Arg Lys Leu Lys Lys Thr Ile Lys Arg Val Leu Lys His 1 5 10 15
Val Pro 32 18 PRT Artificial Synthetic antimicrobial peptide 32 Gly
Leu Phe Ser Leu Leu Arg Lys Thr Ile Lys Lys Val Leu Gln His 1 5 10
15 Ile Arg 33 18 PRT Artificial Synthetic antimicrobial peptide 33
Gly Leu Leu Asn Lys Leu Lys Arg Ala Leu Lys Lys Val Val Lys His 1 5
10 15 Val Arg 34 18 PRT Artificial Synthetic antimicrobial peptide
34 Gly Trp Leu Arg Lys Ile Gly Lys Ala Val Lys Lys Val Val Lys Arg
1 5 10 15 Val Leu 35 18 PRT Artificial Synthetic antimicrobial
peptide 35 Gly Leu Leu Gly Lys Leu Lys Arg Lys Ile Lys Lys Ala Leu
Glu Gly 1 5 10 15 Ile Arg 36 18 PRT Artificial Synthetic
antimicrobial peptide 36 Gly Trp Leu Lys Ile Leu Glu Lys Ala Ala
Lys Ile Thr Val Lys Asn 1 5 10 15 Val Leu 37 18 PRT Artificial
Synthetic antimicrobial peptide 37 Gly Leu Leu Arg Ile Leu Lys Lys
Lys Ala Lys Lys Ala Leu Gln His 1 5 10 15 Ile Leu 38 21 PRT
Artificial Synthetic antimicrobial peptide 38 Gly Leu Leu Gly Lys
Ile Arg Lys Glu Gly Arg Met Phe Trp Arg Val 1 5 10 15 Phe Arg Arg
Trp Leu 20 39 21 PRT Artificial Synthetic antimicrobial peptide 39
Gly Leu Leu Arg Lys Leu Arg Lys Glu Val Lys Lys Val Leu Ser Ile 1 5
10 15 Phe Phe Arg Trp Leu 20 40 21 PRT Artificial Synthetic
antimicrobial peptide 40 Gly Leu Leu Asn Lys Leu Lys Lys Asn Val
Lys Asn Ile Val Gln His 1 5 10 15 Ile Leu Arg Trp Leu 20 41 21 PRT
Artificial Synthetic antimicrobial peptide 41 Gly Leu Phe Ser Ile
Leu Lys Arg Glu Ile Lys Arg Thr Phe Ser Met 1 5 10 15 Phe Tyr Arg
Trp Leu 20 42 21 PRT Artificial Synthetic antimicrobial peptide 42
Gly Leu Leu Gly Lys Leu Lys Lys Glu Leu Lys Asn Val Leu Glu His 1 5
10 15 Ile Tyr Arg Trp Leu 20 43 21 PRT Artificial Synthetic
antimicrobial peptide 43 Gly Ile Leu Ser Lys Leu Lys Lys Ala Ile
Lys Arg Ile Leu Gln Asp 1 5 10 15 Val Leu Arg Trp Leu 20 44 21 PRT
Artificial Synthetic antimicrobial peptide 44 Gly Met Leu Lys Lys
Leu Lys Lys Lys Thr Lys Arg Ala Phe Ser Val 1 5 10 15 Phe Cys Arg
Trp Leu 20 45 21 PRT Artificial Synthetic antimicrobial peptide 45
Gly Ile Leu Ser Met Leu Glu Arg Arg Trp Ser Met Tyr Cys Ser Ile 1 5
10 15 Phe Cys Arg Trp Leu 20 46 21 PRT Artificial Synthetic
antimicrobial peptide 46 Gly Ile Leu Ser Lys Leu Lys Lys Lys Ala
Lys Asn Ala Val Lys Met 1 5 10 15 Phe Cys Arg Trp Leu 20 47 57 DNA
Artificial Synthetic Gen2 gene CDS (1)..(57) 47 ggc ctg ctg agc aaa
ctg aag aag gcg gcg aaa aaa gcg ctg aaa cat 48 Gly Leu Leu Ser Lys
Leu Lys Lys Ala Ala Lys Lys Ala Leu Lys His 1 5 10 15 gtg ctg tag
57 Val Leu 48 18 PRT Artificial Synthetic Construct 48 Gly Leu Leu
Ser Lys Leu Lys Lys Ala Ala Lys Lys Ala Leu Lys His 1 5 10 15 Val
Leu 49 96 DNA Artificial Primer 1 sequence 49 attattcaga tgctggatcc
ggcggaaggc ctgctgagca aactgaagaa ggcggcgaaa 60 aaagcgctga
aacatgtgct gtagctcgag attatt 96 50 96 DNA Artificial Primer 2
sequence 50 aataatctcg agctacagca catgtttcag cgcttttttc gccgccttct
tcagtttgct 60 cagcaggcct tccgccggat ccagcatctg aataat 96 51 20 DNA
Artificial Primer 3 sequence 51 tgctagttat tgctcagcgg 20 52 19 DNA
Artificial Primer 4 sequence 52 accgtagttg cgcccatcg 19
* * * * *